Small-molecule modulators of Trp-p8 activity

ABSTRACT

Provided are small-molecule Trp-p8 modulators, including Trp-p8 agonists and Trp-p8 antagonists, and compositions comprising small-molecule Trp-p8 agonists as well as methods for identifying and characterizing novel small-molecule Trp-p8 modulators and methods for decreasing viability and/or inhibiting growth of Trp-p8 expressing cells, methods for activating Trp-p8-mediated cation influx, methods for stimulating apoptosis and/or necrosis, and related methods for the treatment of diseases, including cancers such as lung, breast, colon, and/or prostate cancers as well as other diseases, such as benign prostatic hyperplasia, that are associated with Trp-p8 expression.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of U.S. Non-Provisional patent application Ser. No. 11/707,546 filed Feb. 15, 2007 which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/773,435 filed on Feb. 15, 2006; the contents of each are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of cell biology, biochemistry, and organic chemistry. More specifically, the present invention provides small-molecule modulators of Trp-p8 activity, which include Trp-p8 agonists and Trp-p8 antagonists, as well as compositions comprising small-molecule Trp-p8 modulators. Also provided are methods for identifying and characterizing novel small-molecule Trp-p8 modulators as well as methods for modulating Trp-p8-mediated cation influx and/or apoptosis in a cell and related methods for the treatment of diseases associated with Trp-p8 expression, activation, and/or signaling. Exemplary diseases suitably treated by the compositions and methods of the present invention include cancers, such as lung, breast, colon, and/or prostate cancers.

BACKGROUND OF THE INVENTION

Prostate carcinoma is the most common cancer diagnosed in men in the United States and has the second highest cancer death rate yielding only to lung adenocarcinoma. Parker et al., CA Cancer J. Clin. 46:5-27 (1996). Although it is possible to effectively treat organ-confined prostate cancer, there are very limited treatment options for metastatic disease. Thus, it is of great importance to find novel ways to diagnose early stage disease and to closely monitor both progression and treatment of the disease, as well as to develop new therapeutic approaches. To achieve this, it is important to understand the molecular mechanisms of prostate cancer development and to identify new biochemical markers for disease diagnosis and progression.

To date there are very few prostate-specific markers available. The best-known and well-characterized markers of proven prostate cancer diagnostic value are the proteins prostatic acid phosphatase (PAP), prostate specific antigen (PSA), and prostate-specific membrane antigen (PSMA). Each of these proteins has also become the target for novel immunotherapy approaches to the treatment of disease. Horoszewicz et al., Anticancer Res. 7:927-935 (1987); Barren et al., Prostate 30:65-68 (1997); Murphy et al., Prostate 33:281-285 (1997); Murphy et al., Prostate 26:164-168 (1995); Rochon et al., Prostate 25:219-223 (1995); Correale et al., J. Immunol. 161:3186-3194 (1998); and Murphy et al., Prostate 38:73-78 (1999).

It has been reported that a cation channel protein, variously designated Trp-p8 (transient receptor potential-p8), TRPM8, and CMR1 (cold and menthol receptor 1), is preferentially expressed in prostate. Cloning of the full-length human Trp-p8 cDNA revealed a transcript corresponding to an 1104 amino acid polypeptide sharing homology with the trp family of calcium channels. Clapham et al., Nature Reviews 2:387-396 (2001) and Clapham et al., IUPHAR Compendium, TRP Channels (2002). Trp-p8 shows particularly high homology with the human TRPC7 gene—a putative Ca²⁺ channel protein of the trp family that is highly expressed in brain tissue. Nagamine et al., Genomics 54:124-131 (1998). Trp-p8 also shows significant homology to human melastatin, another Trp family-related protein expressed in melanocytes and believed to be a tumor suppressor gene. Duncan et al., Cancer Res. 58:1515-1520 (1998) and Hunter et al., Genomics 54:116-123 (1998). Perhaps of greatest interest is the observation that the Trp-p8 gene appears to be expressed in a large spectrum of nonprostatic, in addition to prostatic, neoplastic lesions. Tsavaler et al., Cancer Res. 61(9):3760-9 (2001).

The Trp superfamily comprises more than 20 related cation channel proteins that have been implicated in processes including sensory physiology to vasorelaxation and male fertility. Defects in Trp channels have been associated with changes in growth control and tumor suppression. While all Trp proteins are calcium channels, they vary significantly in their selectivity and mode of activation. Members of the Trp superfamily share significant sequence homology and predicted structural similarities, such as size of predicted transmembrane segments.

Trp-p8 is over-expressed in a range of cancers including prostate, breast, lung and colon, while within normal tissues, it is predominantly expressed in human prostate [Tsavaler et al., supra] and dorsal root ganglia (DRG), (Dendreon, unpublished observation). Fuessel et al. reported that Trp-p8 is a highly prostate-specific and prostate carcinoma-associated gene thus qualifying it as a potential target for specific therapies. International J. of Oncology 23:221-228 (2003). Among other species, Trp-p8 orthologues are reportedly expressed in a subset of DRG and trigerminal ganglia (TG) neurons in rat [McKemy et al., Nature 416(6876):52-8 (2002)] and mouse [Peier et al., Cell 108(5):705-15 (2002)] as well. Thus, Trp-p8 is a pantumor-expressed marker with significant potential use in disease diagnosis and monitoring of disease progression during treatment as well as a viable target for cancer therapy.

Association of Trp-p8 with prostate, lung, breast, and colon cancers and the important role various ion channels play in vital cell functions suggest that the Trp-p8 channel may have a significant function in cancer cell signaling and/or proliferation. Modulation of Trp-p8 activity, either by activating via an agonist or inhibiting via an antagonist, at a physiological temperature can be valuable as a therapeutic to manipulate the Trp-p8 expressing cells in a specific manner. See for example U.S. patent application Ser. No. 10/923,413.

Accordingly, there remains a need in the art for small-molecule modulators of Trp-p8 activity, compositions comprising one or more small-molecule Trp-p8 modulators, and methods for the identification and use of small-molecules for modulating the activity of Trp-p8 in a cell and for the treatment of disease associated with the aberrant expression of Trp-p8.

BRIEF SUMMARY OF THE INVENTION

The present invention fulfills these and other related needs by providing small molecule modulators of Trp-p8 activity, including Trp-p8 agonists and Trp-p8 antagonists, as well as compositions comprising such Trp-p8 modulators, and methods for identifying and using Trp-p8 modulators. Within certain embodiments, compounds of the present invention bind to and activate Trp-p8 and/or stimulate cation influx, including but not limited to calcium influx, in a cell wherein cation influx is correlative of Trp-p8 modulator induced toxicity. Thus, within these and other embodiments, Trp-p8 agonists of the present invention are effective in inhibiting growth of and/or inducing apoptosis and/or necrosis in a cell expressing Trp-p8. Within alternative embodiments are provided Trp-p8 antagonists that are effective in reducing the basal activity of Trp-p8 in a cell thereby reducing the viability of Trp-p8 expressing cells. Advantageously, therefore, agonists and antagonists of the present invention can be used to treat diseases including, but not limited to, cancers of the breast, lung, colon, and/or prostate, that are associated with Trp-p8 expression.

One or more Trp-p8 modulator can be formulated in compositions, including pharmaceutical compositions, comprising one or more pharmaceutically acceptable carrier or excipient and/or one or more additional therapeutic compound. Such compositions will find utility in methods for the treatment of one or more disease associated with Trp-p8 expression.

Thus, in one embodiment, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I:

wherein

R₁ is selected from H, alkyl, heteroalkyl, arylalkyl, and aryl, or, R₁ and R₂ together with the nitrogen group may form a cyclic or heterocyclic group of up to 25 atoms;

R₂ is selected from aryl and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-A:

wherein A, B, C, and D are independently selected from CR₂ and N; wherein at least one of A, B, C, and D is CR₂; wherein R₂ is a member selected from H, alkyl, heteroalkyl, aryl, halogen, and arylalkyl, R₆O—, and R₆S—, wherein R₆ is alkyl; wherein when two adjacent of A, B, C, and D are CR₂, the two R₂'s may combine to form a single aryl, cycloalkyl, or heterocycloalkyl group; and

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, aryl, arylalkyl, —NR₇C(O)—, —C(O)NR₇—, —O—, —S—, —S(O)—, —S(O)₂—, and —NR₇—, wherein R₇ is a member selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₄ is selected from —C(O)R₈—, alkyl, arylalkyl, and heteroalkyl, wherein R₈ is selected from alkyl and heteroalkyl;

R₅ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₄ and R₅ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-A, R₁ is H; R₇ is H; R₈ comprises 2, 3, or 4 carbons; R₄ is selected from propionyl, ethyl, butyryl, hydroxypropionyl, and 3-hydroxybutyryl; R₅ is selected from H and methyl; R₆ comprises 1, 2, 3, 4, 5, or 6 carbons; and/or R₂ is selected from methoxy, methylsulfanyl, phenyl, and H.

Exemplified herein are compounds of Formula I-A comprising a group selected from 2-(2-amino-propionylamino)-4-methoxy-phenyl, N-(2-Amino-ethyl)-2-amino-5-methylsulfanyl-phenyl, 1-(2-amino-ethoxy)-naphthalen-2-yl, 2-(2-amino-ethylamino)-4-methylsulfanyl-phenyl, N-(2-Amino-ethyl)-5-methoxy-benzamide, 2-(2-amino-butyrylamino)-4-methoxy-phenyl, 2-(2-amino-3-hydroxy-propionylamino)-4-methoxy-phenyl, 3-(2-amino-ethylamino)-naphthalen-2-yl, N-(2-Amino-ethyl)-2-amino-benzamide, 2-(2-amino-3-hydroxy-propionylamino)-4-methoxy-phenyl, 2-(2-amino-acetylamino)-phenyl, 2-(2-amino-3-hydroxy-butyrylamino)-4-methoxy-phenylamide, and 2-(2-amino-acetylamino)-4-methoxy-phenyl.

Within alternative related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-B:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, alkyl, heteroalkyl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-B, R₁ is H; R₃ is selected from methylene, ethylene, propylene, and butylene; R₄ is selected from H and methyl; and/or R₂ is selected from phenyl, furan, methylpyrrole, methylbenzoate, aminophenyl, hydroxyphenyl, cyanophenyl, and methoxyphenyl.

Exemplified herein are compounds of Formula I-B comprising a group selected from 2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-amino-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-hydroxy-phenyl)-2H-pyrazol-3-yl, 2-(2-methylamino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-cyano-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-methoxy-phenyl)-2H-pyrazol-3-yl, 4-{1-(2-Amino-ethyl)-1H-pyrazol-3-yl}-benzoic acid methyl ester, 2-(2-amino-ethyl)-5-(3-amino-phenyl)-2H-pyrazol-3-yl, and 2-(2-amino-ethyl)-5-(3-hydroxy-phenyl)-2H-pyrazol-3-yl.

Within still further related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-C:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, arylalkyl, —NHC(O)R₅—, —OR₅—, and —NHR₅—, wherein R₅ is alkyl or heteroalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-C, R₁ is H; R₂ is phenyl; R₅ is selected from, methylene, ethylene, propylene, and butylene; R₃ is selected from propionylamino, ethoxy, propoxy, and ethylamino; and/or R₄ is selected from H and methyl.

Exemplified herein are compounds of Formula I-C comprising a group selected from 2-(2-amino-propionylamino)-2-phenyl-ethyl, 2-(2-amino-ethoxy)-2-phenyl-ethyl, 2-(2-amino-ethoxy)-2-phenyl-ethyl, 2-(3-amino-propoxy)-2-phenyl-ethyl, 2-(2-dimethylamino-ethoxy)-2-phenyl-ethyl, and 2-(2-amino-ethylamino)-2-phenyl-ethyl.

Within still further related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-D:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-D, R₁ is H; R₂ is selected from phenyl and phenylamino; R₃ is selected from methylene, ethylene, propylene, butylene, methylamino, ethylamino, propylamino, butylamino, and acetyl; and/or R₄ is selected from H and methyl.

Exemplified herein are compounds of Formula I-D comprising a group selected from 2-[2-(2-amino-ethylamino)-phenyl]-ethyl, 2-(2-aminomethyl-phenyl)-ethyl, and 2-[(2-amino-acetyl)-phenyl-amino]-ethyl.

Within yet other related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-E:

wherein A, B, C, and D are independently selected from CR₁ and N; wherein at least one of A, B, C, and D is CR₁; wherein R₁ is selected from H, alkyl, heteroalkyl, aryl, arylalkyl and halogen; wherein when two adjacent of A, B, C, and D are CR₁, the two R₁'s may combine to form a single aryl, cycloalkyl, or heterocycloalkyl group;

R₂ is selected from alkyl, heteroalkyl and arylalkyl;

R₃ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₂ and R₃ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-E,

(i) R₁ is H or —OR^(i) and R^(i) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(ii) R₁ is —SR^(ii). and wherein R^(ii) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(iii) R₁ is —S(O)R^(iii) and wherein R^(iii) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

iv) R₁ is —S(O)₂R^(iv) and wherein R^(iv) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(v) R₁ is —C(O)NR^(v)R^(vi), wherein R^(v) and R^(vi) are independently selected from H, methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, diethylaminoethyl, phenyl, pyridinyl, methoxyethyl, hydroxyethoxyethyl, benzyl, methylphenyl, phenylethyl, hydroxyhydroxymethylphenylethyl, carbamoylmethyl, and hydroxymethyl hydroxyethyl;

(vi) R₁ is —C(O)NR^(v)R^(vi), wherein R^(v) and R^(vi) together form morpholine, piperazine, piperazine ethyl ester;

(vii) R₂ is selected from methylene, ethylene, propylene, and butylene;

(viii) R₂ is ethylene and R₃ is H; and

(ix) R₁ is CF₃ or halogen.

Exemplified herein are compounds of Formula I-E comprising a group selected from 3-(2-amino-ethyl)-5-methoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(3-hydroxy-propoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-ethoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-methanesulfonyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(2-hydroxy-ethoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid amide, 3-(2-Amino-ethyl)-5-methylsulfanyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-methanesulfinyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-diethylamino-ethyl)-amide, 3-(2-Amino-propyl)-2,3-dihydro-benzoimidazol-2-one, [3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazol-5-yloxy]-acetonitrile, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid ethylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid pyridin-3-ylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-methoxy-ethyl)-amide, 1-(2-Amino-ethyl)-1,3-dihydro-benzoimidazol-2-one, 1-(2-Amino-ethyl)-1,3-dihydro-naphtho[2,3-d]imidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethyl)-amide, 3-(2-Amino-ethyl)-5-propoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-c]pyridin-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-4-carboxylic acid (2-diethylamino-ethyl)-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid pyridin-4-ylamide, 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-b]pyridin-2-one, 1-(3-Amino-propyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid phenylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid [2-(2-hydroxy-ethoxy)-ethyl]-amide, 1-(2-Amino-ethyl)-5-trifluoromethyl-1,3-dihydro-benzoimidazol-2-one, 1-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-c]pyridin-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid benzylamide, 3-(2-Amino-ethyl)-5-(morpholine-4-carbonyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(2-oxo-2-phenyl-ethoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-methylamino-ethyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-butoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid methyl-phenyl-amide, 4-[3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carbonyl]-piperazine-1-carboxylic acid ethyl ester, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid diethylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid phenethyl-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-1-hydroxymethyl-2-phenyl-ethyl)-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid carbamoylmethyl-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-1-hydroxymethyl-ethyl)-amide, N-{2-[2-oxo-2,3-dihydro-benzoimidazol-1-yl]-ethyl}-guanidine, 3-(2-Amino-ethyl)-5-benzyloxy-1,3-dihydro-benzoimidazol-2-one, and 1-(4-Amino-butyl)-1,3-dihydro-benzoimidazol-2-one. Within one such embodiment, is provided the compound 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methoxy-1,3-dihydro-benzoimidazol-2-one.

Other aspects of the present invention provide compositions, including pharmaceutical compositions, comprising one or more small-molecule Trp-p8 modulators of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E in combination with a pharmaceutically acceptable excipient, carrier and/or diluent. Exemplified herein within the Examples are specific Trp-p8 agonists and antagonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E; methods for synthesizing exemplary Trp-p8 agonists and antagonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E; and EC50 data demonstrating the in vitro efficacy and specific activity of each of the disclosed Trp-p8 agonists and antagonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E.

Within still further aspects, compositions of the present invention comprise one or more compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E formulated together with one or more cancer therapeutic agent. Alternatively, compositions of the present invention comprise a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E independently formulated with one or more cancer therapeutic agent. That is, one or more compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E and the cancer therapeutic agent are separately formulated.

Suitable cancer therapeutic agents include, but are not limited to, antimitotic agents including, but not limited to, paclitaxel, vincristine, and etoposide; alkylating agents including, but not limited to, mechlorethamine, cyclophosphamide, and carmustine; antimetabolites including, but not limited to, methotrexate, gemcitabine, lometrexol, 5-fluorouracil, and 6-mercaptopurine; cytotoxic antibiotics including, but not limited to, doxorubicin, daunorubicin, bleomycin, mitomycin C, and streptozocin; platinum agents including, but not limited to, cisplatin and carboplatin; hormonal agents including, but not limited to, anti-estrogens such as tamoxifen and diethylstilbestrol as well as anti-androgens such as flutamide; antiangiogenesis agents; and farnesyl transferase inhibitors.

In certain aspects, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are administered in combination with cancer therapeutic agents that are themselves ineffective for modulating Trp-p8 activity in a cell expressing Trp-p8. Surprisingly, these types of combination therapies result in enhanced efficacy relative to the use of a single compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E alone.

In other aspects, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are administered in combination with one or more additional Trp-p8 modulator(s) including, but not limited to, a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E.

Within certain of these embodiments are provided small-molecule antagonists of the small-molecule Trp-p8 agonists presented herein. Thus, within certain embodiments are provided small-molecule Trp-p8 antagonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E, and derivatives thereof, of one or more Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E Trp-p8 agonist.

Further embodiments of the present invention provide methods for decreasing cell viability and/or inhibiting cell growth, methods for stimulating cation influx, and methods for inducing apoptosis and/or necrosis in a cell expressing Trp-p8. Exemplary such methods comprise the step of contacting a cell with a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E in a concentration and for a time required to decrease cell viability and/or inhibit cell growth, to raise intracellular calcium, and/or to induce apoptosis and/or necrosis of the cell.

In still further embodiments, the present invention provides methods for treating a disease in a mammal, most typically a human, by administering one or more compound and/or composition of the present invention. In certain aspects, the methods include the administration of a composition comprising a combination of a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E with one or more cancer therapeutic agent delivered in a simultaneous manner, such as in a single formulation. In certain other aspects, the methods of the present invention include combination therapy wherein the compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E is administered first in one formulation, followed by the cancer therapeutic agent in a separate formulation. The methods also include a cancer therapeutic agent being delivered first in one formulation, followed by a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E in a separate formulation.

Therapeutic methods of the present invention are particularly effective in the treatment of cancers associated with the expression of Trp-p8 including, but not limited to, certain colon, lung, breast, and prostate cancers.

The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description, read in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B are graphs depicting an exemplary ATP viability assay suitable for testing and characterizing small-molecule Trp-p8 modulators of the present invention. In a preliminary assay (FIG. 1A), compounds were tested at 1 μM and specific killing of Trp-p8 expressing CHO cells (CHO/Trp-p8) measured at 37° C. In a follow up assay (FIG. 1B), compounds were tested at various concentrations, and killing of Trp-p8 expressing CHO cells (CHO/Trp-p8) was measured at 37° C. An EC₅₀ value was derived from a plot of cell viability as a function of concentration

FIGS. 2A-2C are graphs depicting Trp-p8 modulator-induced increases in intracellular calcium as determined by a calcium flux assay performed at 37° C. FIG. 2A is a positive control demonstrating that CHO and CHO/Trp-p8 cells respond similarly to 2 μM Ionomycin at 37° C. in the calcium flux assay. FIG. 2B is a negative control demonstrating that parental CHO cells that do not express endogenous or exogenous Trp-p8 do not respond to Trp-p8 agonists at a concentration of 10 μM. FIG. 2C demonstrates that a Trp-p8 agonist induced a specific, concentration-dependent response in CHO/Trp-p8 cells at 37° C.

FIG. 3 are plots of flow cytometry data demonstrating that a Trp-p8 agonist is capable of specifically inducing apoptosis in Trp-p8 expressing CHO cells at 37° C., in a dose-dependent manner.

FIG. 4 is a graph depicting exemplary results from a primary screen for Trp-p8 antagonists using the ATP viability assay, described herein, with CHO/Trp-p8 cells at 37° C. CHO/Trp-p8 cells were exposed to compounds, at different concentrations, in 1% DMSO or 1% DMSO in combination with a toxic concentration of a Trp-p8 agonist. The viability of cells was measured after 24-26 hours at 37° C. using the ATP assay. Compounds that protected the cells from the toxic effect of the Trp-p8 agonist are classified as Trp-p8 antagonists (Compounds A-B). An inactive compound (Compound C) had no protective effect and is shown here for the purpose of illustration of the assay.

FIG. 5 is a graph depicting the screening and characterization of Trp-p8 antagonists by the calcium flux assay performed at 37° C. CHO/Trp-p8 cells were loaded with the calcium indicator dye, Fura-2, and the increase in intracellular calcium in response to compounds was determined by the increase in fluorescence. Fura-2 dye loaded CHO/Trp-p8 cells were exposed to 1% DMSO or an antagonist, at different concentrations, in 1% DMSO at 37° C. Three minutes later, an agonist was added to the cells. When cells were exposed to effective concentrations of the antagonist, their ability to respond to the agonist was significantly reduced or eliminated altogether.

FIGS. 6A-6B are graphs depicting results from an exemplary animal model suitable for testing and characterizing small-molecule Trp-p8 modulators of the present invention. Mice were injected subcutaneously with CHO/Trp-p8 cells, resulting in the formation of solid tumors. The length (longest dimension) and width (dimension perpendicular to and in the same plane as length) of each tumor was measured with Vernier calipers, and the tumor volume was approximated by a formula for the volume of an ellipsoid: 0.52*L*W². When the average tumor volume reached approximately 100 mm³, the mice were randomized into groups. In FIG. 6A, the mice were administered a single dose of either a study compound as an aqueous formulation, or vehicle alone, by oral gavage. In FIG. 6B, the mice were dosed repeatedly with either a study compound as an aqueous formulation, or vehicle alone, by oral gavage. Tumors were then subsequently measured on the indicated days. The data is presented as mean tumor volumes±standard error of the mean.

FIG. 7 is a graph depicting the plasma concentrations of several compounds, as a function of time and dose, in mice after administration via a single intraperitoneal injection. The compounds were all dissolved in an aqueous formulation, and administered at comparable dose levels. Blood was collected at the indicated time points, and analyzed for drug levels.

FIGS. 8A-8B are graphs depicting the efficacy demonstrated by several compounds in a murine tumor xenograft model expressing Trp-p8. Mice were injected subcutaneously with CHO/Trp-p8 cells, resulting in the formation of solid tumors. The length (L; longest dimension) and width (W; dimension perpendicular to and in the same plane as length) of each tumor was measured with calipers, and the tumor volume was approximated by a formula for the volume of an ellipsoid: 0.52*L*W². When the mean tumor volume reached approximately 100 mm³, the mice were randomized into groups, and administered either a compound as an aqueous formulation, or vehicle alone, by intraperitoneal injection, on the indicated days. All compounds were administered at comparable dose levels. Tumors were subsequently measured on the indicated days. The data is presented as mean tumor volumes±standard error of the mean.

FIGS. 9A and 9B are graphs depicting the plasma concentrations of several compounds, as a function of time and dose, in rats (FIG. 9A), and dogs (FIG. 9B) after a single oral dose. The compounds were all dissolved in an aqueous formulation, and administered at comparable dose levels by oral gavage. Blood was collected at the indicated time points, and analyzed for drug levels.

FIGS. 10A and 10B are graphs depicting the efficacy demonstrated by several compounds in a murine tumor xenograft model expressing Trp-p8. Mice were injected subcutaneously with CHO/Trp-p8 cells, resulting in the formation of solid tumors. The length (L; longest dimension) and width (W; dimension perpendicular to and in the same plane as length) of each tumor was measured with Vernier calipers, and the tumor volume was approximated by a formula for the volume of an ellipsoid: 0.52*L*W². When the mean tumor volume reached approximately 100 mm³, the mice were randomized into groups, and administered a single dose of either a compound as an aqueous formulation, or vehicle alone, by oral gavage. Tumors were then subsequently measured on the indicated days. The data is presented as mean tumor volumes±standard error of the mean.

FIG. 11 is a graph depicting the lack of efficacy demonstrated by an exemplary compound in a murine tumor xenograft model lacking Trp-p8 expression. Mice were injected subcutaneously with CHO-K1 cells lacking Trp-p8, resulting in the formation of solid tumors. The length (L; longest dimension) and width (W; dimension perpendicular to and in the same plane as length) of each tumor was measured with Vernier calipers, and the tumor volume was approximated by a formula for the volume of an ellipsoid: 0.52*L*W². When the average tumor volume reached approximately 50 mm³, the mice were randomized into groups, and administered either a single dose of the compound as an aqueous formulation, or vehicle, by oral gavage. Tumors were then subsequently measured on the indicated days. The data is presented as mean tumor volumes±standard error of the mean.

FIG. 12 is a graph depicting the efficacy demonstrated by and exemplary compound in a murine tumor xenograft model expressing Trp-p8. The LuCaP tumor model was obtained from Robert L. Vassella, Ph.D., Professor of Urology in the University of Washington's School of Medicine. The length (L; longest dimension) and width (W; dimension perpendicular to and in the same plane as length) of each tumor was measured with Vernier calipers, and the tumor volume was approximated by a formula for the volume of an ellipsoid: 0.52*L*W². When the average tumor volume reached approximately 150 mm³, the mice were administered the compound, as an aqueous formulation, by intraperitoneal injection, once a day for 5 days. Tumors were then subsequently measured on the indicated days. The data is presented as mean tumor volumes±standard error of the mean.

SEQ ID NO: 1 is the nucleotide sequence of a human Trp-p8 cDNA (GenBank Accession No. AY090109).

SEQ ID NO: 2 is the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 (GenBank Accession No. NP_(—)076985).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that certain small-molecule Trp-p8 modulators, including agonists of Trp-p8 activity, are capable of inhibiting the growth of and/or inducing apoptosis and/or necrosis in cells that express Trp-p8. Without wishing to be limited to any specific mode of action, it is believed that Trp-p8 agonist-mediated activation of the Trp-p8 receptor substantially increases cation influx, which is correlative of cellular toxicity. It is further believed that Trp-p8 antagonists can inhibit the basal level and/or native ligand-induced activity of endogenous Trp-p8 activation which, consequently, leads to reduced growth or death of cells expressing this cation channel protein.

Thus, the present invention provides small-molecule Trp-p8 modulators, including agonists and antagonists of Trp-p8 activity, as well as compositions, including pharmaceutical compositions, comprising one or more small-molecule Trp-p8 modulator in combination with one or more pharmaceutically acceptable carrier and/or excipient. The present invention also provides combination compositions comprising one or more Trp-p8 modulator and one or more additional therapeutic compound such as, for example, a cancer therapeutic agent. Trp-p8 modulators and compositions comprising Trp-p8 modulators will find utility in methods for activating Trp-p8-mediated cation influx in a cell, methods for inducing apoptosis and/or necrosis in a cell, as well as methods for the treatment of diseases associated with Trp-p8 expression including, but not limited to, cancers, such as breast, colon, lung, and prostate cancers.

DEFINITIONS

The term “Trp-p8 modulators” refers collectively to compounds which are small-molecule agonists and antagonists that bind to and either increase or decrease, respectively, the activity of Trp-p8 in a cell. Trp-p8 agonists include compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E and chemical derivatives thereof. Trp-p8 antagonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E may be readily synthesized and characterized by a skilled artisan by employing the methodology expressly provided herein and/or as is readily available in the art.

The phrase “activate Trp-p8” means agonist-mediated activation of Trp-p8 expressed on the surface of a cell. For example, within certain embodiments, agonists of the present invention, when contacted with a cell and/or administered in vivo to a mammalian subject, activate Trp-p8 thereby facilitating the influx of cations, such as calcium ions, to such an intracellular level and/or for such a duration that is sufficient to cause toxicity to the cell as evidenced by a diminution in cell growth and/or an onset of necrotic and/or apoptotic cell death.

The term “aliphatic amine” means a substituted nitrogen atom wherein any substituents, other than H, are attached to the nitrogen by a saturated carbon atom.

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon group, or combination thereof, which may be fully saturated, mono or polyunsaturated and can include di and multivalent groups, having the number of carbon atoms designated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbon groups include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)ethyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

The term “alkenyl” denotes branched or unbranched hydrocarbon chains containing one or more carbon-carbon double bonds.

The term “alkynyl” refers to branched or unbranched hydrocarbon chains containing one or more carbon-carbon triple bonds.

The term “alkylene” by itself or as part of another substituent means a divalent group derived from an alkane, as exemplified by —CH₂CH₂CH₂CH₂—. Typically, an alkylene group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

The term “cycloalkylene” by itself or as part of another substituent means a divalent group derived from a cycloalkane, as exemplified by cyclohexylene. Typically, a cycloalkylene group will have from 5-8 carbon atoms, with those groups having 6 carbon atoms being preferred in the present invention.

The term “alkenylene” by itself or as part of another substituent means a divalent group derived from an alkenyl, as exemplified by —CH═CHCH₂CH₂—. Typically, alkenylene groups will have from 2 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.

The terms “alkoxy,” “alkylamino” and “alkylthio” refer to those groups having an alkyl group attached to the remainder of the molecule through an oxygen, nitrogen or sulfur atom, respectively. Similarly, the term “dialkylamino” is used in a conventional sense to refer to —NR′R″ wherein the R groups can be the same or different alkyl groups.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon, or combinations thereof, fully saturated or containing from 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃—)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃—, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term “heteroalkyl” are those groups described in more detail below as “heterocycloalkyl.” The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

The term “acyl” refers to those groups derived from an organic acid by removal of the hydroxy portion of the acid. Accordingly, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.

An “activated carbonyl” group is a carbonyl group whose electrophilicity is enhanced as a result of the groups attached to either side of the carbonyl. Examples of such activated carbonyl groups are (polyfluoroalkyl)ketones, (polyfluoroalkyl)aldehydes, alpha-keto esters, alpha-keto acids, alpha-keto amides, 1,2-diketones, 2-acylthiazoles, 2-acylimidazoles, and the like.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “fluoroalkyl,” are meant to include monofluoroalkyl and polyfluoroalkyl.

The term “aryl,” employed alone or in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated, an aromatic substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.

The term “heteroaryl” is meant to include those aryl rings which contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. The “heteroaryl” groups can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl ring systems are selected from the group of acceptable substituents described below. The term “arylalkyl” is meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkyl group (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”) are meant to include both substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of group are provided below.

Substituents for the alkyl and heteroalkyl groups (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2N+1), where N is the total number of carbon atoms in such group. R′, R″ and R′ each independently refer to hydrogen, unsubstituted(C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkyl groups. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similarly, substituents for the aryl groups are varied and are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, —NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR″—S(O)₂—R′, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″ and R′″ are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂— or a single bond, and the subscript q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), and sulfur (S).

The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino, magnesium salt, or other similar salt. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrophosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.

Small-Molecule Modulators of Trp-p8 Activity

Small-molecule Trp-p8 modulators that are suitably employed in the compositions and methods of the present invention are exemplified herein by compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E disclosed herein, and derivatives thereof.

Thus, in one embodiment, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I:

wherein

R₁ is selected from H, alkyl, heteroalkyl, arylalkyl, and aryl, or, R₁ and R₂ together with the nitrogen group may form a cyclic or heterocyclic group of up to 25 atoms;

R₂ is selected from aryl and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-A:

wherein A, B, C, and D are independently selected from CR₂ and N; wherein at least one of A, B, C, and D is CR₂; wherein R₂ is a member selected from H, alkyl, heteroalkyl, aryl, halogen, arylalkyl, R₆O—, and R₆S—, wherein R₆ is alkyl; wherein when two adjacent of A, B, C, and D are CR₂, the two R₂'s may combine to form a single aryl, cycloalkyl, or heterocycloalkyl group; and

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, aryl, arylalkyl, —NR₇C(O)—, —C(O)NR₇—, —O—, —S—, —S(O)—, —S(O)₂—, and —NR₇—, wherein R₇ is a member selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₄ is selected from —C(O)R₈—, alkyl, arylalkyl, and heteroalkyl, wherein R₈ is selected from alkyl and heteroalkyl;

R₅ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₄ and R₅ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-A, R₁ is H; R₇ is H; R₈ comprises 2, 3, or 4 carbons; R₄ is selected from propionyl, ethyl, butyryl, hydroxypropionyl, and 3-hydroxybutyryl; R₅ is selected from H and methyl; R₆ comprises 1, 2, 3, 4, 5, or 6 carbons; and/or R₂ is selected from methoxy, methylsulfanyl, phenyl, and H.

Exemplified herein are compounds of Formula I-A comprising a group selected from 2-(2-amino-propionylamino)-4-methoxy-phenyl, N-(2-Amino-ethyl)-2-amino-5-methylsulfanyl-phenyl, 1-(2-amino-ethoxy)-naphthalen-2-yl, 2-(2-amino-ethylamino)-4-methylsulfanyl-phenyl, N-(2-Amino-ethyl)-5-methoxy-benzamide, 2-(2-amino-butyrylamino)-4-methoxy-phenyl, 2-(2-amino-3-hydroxy-propionylamino)-4-methoxy-phenyl, 3-(2-amino-ethylamino)-naphthalen-2-yl, N-(2-Amino-ethyl)-2-amino-benzamide, 2-(2-amino-3-hydroxy-propionylamino)-4-methoxy-phenyl, 2-(2-amino-acetylamino)-phenyl, 2-(2-amino-3-hydroxy-butyrylamino)-4-methoxy-phenylamide, and 2-(2-amino-acetylamino)-4-methoxy-phenyl.

Within alternative related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-B:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, alkyl, heteroalkyl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-B, R₁ is H; R₃ is selected from methylene, ethylene, propylene, and butylene; R₄ is selected from H and methyl; and/or R₂ is selected from phenyl, furan, methylpyrrole, methylbenzoate, aminophenyl, hydroxyphenyl, cyanophenyl, and methoxyphenyl.

Exemplified herein are compounds of Formula I-B comprising a group selected from 2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-amino-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-hydroxy-phenyl)-2H-pyrazol-3-yl, 2-(2-methylamino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-cyano-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-methoxy-phenyl)-2H-pyrazol-3-yl, 4-{1-(2-Amino-ethyl)-1H-pyrazol-3-yl}-benzoic acid methyl ester, 2-(2-amino-ethyl)-5-(3-amino-phenyl)-2H-pyrazol-3-yl, and 2-(2-amino-ethyl)-5-(3-hydroxy-phenyl)-2H-pyrazol-3-yl.

Within still further related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-C:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, arylalkyl, —NHC(O)R₅—, —OR₅—, and —NHR₅—, wherein R₅ is alkyl or heteroalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-C, R₁ is H; R₂ is phenyl; R₅ is selected from, methylene, ethylene, propylene, and butylene; R₃ is selected from propionylamino, ethoxy, propoxy, and ethylamino; and/or R₄ is selected from H and methyl.

Exemplified herein are compounds of Formula I-C comprising a group selected from 2-(2-amino-propionylamino)-2-phenyl-ethyl, 2-(2-amino-ethoxy)-2-phenyl-ethyl, 2-(2-amino-ethoxy)-2-phenyl-ethyl, 2-(3-amino-propoxy)-2-phenyl-ethyl, 2-(2-dimethylamino-ethoxy)-2-phenyl-ethyl, and 2-(2-amino-ethylamino)-2-phenyl-ethyl.

Within still further related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-D:

wherein

R₁ is selected from H, alkyl, heteroalkyl, aryl, and arylalkyl;

R₂ is selected from aryl, and arylalkyl;

R₃ is selected from alkyl, heteroalkyl, and arylalkyl;

R₄ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₃ and R₄ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-D, R₁ is H; R₂ is selected from phenyl and phenylamino; R₃ is selected from methylene, ethylene, propylene, butylene, methylamino, ethylamino, propylamino, butylamino, and acetyl; and/or R₄ is selected from H and methyl.

Exemplified herein are compounds of Formula I-D comprising a group selected from 2-[2-(2-amino-ethylamino)-phenyl]-ethyl, 2-(2-aminomethyl-phenyl)-ethyl, and 2-[(2-amino-acetyl)-phenyl-amino]-ethyl.

Within yet other related embodiments, the present invention provides small-molecule Trp-p8 modulators and derivatives thereof wherein the small-molecules include compounds of the following Formula I-E:

wherein A, B, C, and D are independently selected from CR₁ and N; wherein at least one of A, B, C, and D is CR₁; wherein R₁ is selected from H, alkyl, heteroalkyl, aryl, arylalkyl, and halogen; wherein when two adjacent of A, B, C, and D are CR₁, the two R₁s may combine to form a single aryl, cycloalkyl, or heterocycloalkyl group;

R₂ is selected from alkyl, heteroalkyl and arylalkyl;

R₃ is selected from H, alkyl, heteroalkyl, and arylalkyl; and

R₂ and R₃ together with the nitrogen group form an aliphatic amine.

Within certain exemplary compounds of Formula I-E,

(i) R₁ is H or —OR^(i) and R^(i) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(ii) R₁ is —SR^(ii) and wherein R^(ii) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(iii) R₁ is —S(O)R^(iii) and wherein R^(iii) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

(iv) R₁ is —S(O)₂R^(iv) and wherein R^(iv) is selected from methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, acetonitrile, phenyl, phenylmethoxy, phenylethoxy, phenylpropoxy, phenylbutoxy, and benzyl;

v) R₁ is —C(O)NR^(v)R^(vi), wherein R^(v) and R^(vi) are independently selected from H, methyl, hydroxymethyl, ethyl, hydroxyethyl, propyl, hydroxypropyl, butyl, hydroxybutyl, diethylaminoethyl, phenyl, pyridinyl, methoxyethyl, hydroxyethoxyethyl, benzyl, methylphenyl, phenylethyl, hydroxyhydroxymethylphenylethyl, carbamoylmethyl, and hydroxymethyl hydroxyethyl;

(vi) R₁ is —C(O)NR^(v)R^(vi), wherein R^(v) and R^(vi) together form morpholine, piperazine, piperazine ethyl ester;

(vii) R₂ is selected from methylene, ethylene, propylene, and butylene;

(viii) R₂ is ethylene and R₃ is H

(ix) R₁ is CF₃ or halogen.

Exemplified herein are compounds of Formula I-E comprising a group selected from 3-(2-amino-ethyl)-5-methoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(3-hydroxy-propoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-ethoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-methanesulfonyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(2-hydroxy-ethoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid amide, 3-(2-Amino-ethyl)-5-methylsulfanyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-methanesulfinyl-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-diethylamino-ethyl)-amide, 3-(2-Amino-propyl)-2,3-dihydro-benzoimidazol-2-one, [3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazol-5-yloxy]-acetonitrile, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid ethylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid pyridin-3-ylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-methoxy-ethyl)-amide, 1-(2-Amino-ethyl)-1,3-dihydro-benzoimidazol-2-one, 1-(2-Amino-ethyl)-1,3-dihydro-naphtho[2,3-d]imidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethyl)-amide, 3-(2-Amino-ethyl)-5-propoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-c]pyridin-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-4-carboxylic acid (2-diethylamino-ethyl)-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid pyridin-4-ylamide, 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-b]pyridin-2-one, 1-(3-Amino-propyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid phenylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid [2-(2-hydroxy-ethoxy)-ethyl]-amide, 1-(2-Amino-ethyl)-5-trifluoromethyl-1,3-dihydro-benzoimidazol-2-one, 1-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5-c]pyridin-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid benzylamide, 3-(2-Amino-ethyl)-5-(morpholine-4-carbonyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-(2-oxo-2-phenyl-ethoxy)-1,3-dihydro-benzoimidazol-2-one, 3-(2-methylamino-ethyl)-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-5-butoxy-1,3-dihydro-benzoimidazol-2-one, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid methyl-phenyl-amide, 4-[3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carbonyl]-piperazine-1-carboxylic acid ethyl ester, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid diethylamide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid phenethyl-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-1-hydroxymethyl-2-phenyl-ethyl)-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid carbamoylmethyl-amide, 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-hydroxy-1-hydroxymethyl-ethyl)-amide, N-{2-[2-oxo-2,3-dihydro-benzoimidazol-1-yl]-ethyl}-guanidine, 3-(2-Amino-ethyl)-5-benzyloxy-1,3-dihydro-benzoimidazol-2-one, and 1-(4-Amino-butyl)-1,3-dihydro-benzoimidazol-2-one. Within one such embodiment, is provided the compound 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methoxy-1,3-dihydro-benzoimidazol-2-one.

Synthesis of Small-Molecule Trp-p8 Modulators

As noted above, compounds of the present invention include compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E. Within certain aspects, compounds can be made using commercially available starting materials by employing synthetic methodology readily available in the art. Compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E may be isolated using typical isolation and purification techniques known in the art, including, for example, chromatographic and recrystallization methods.

Those of skill in the art will readily recognize that compounds suitably included in the compositions and methods of the present invention can exist in a number of cis and trans isomers, E/Z forms, diastereomers, as well as optical isomers. Thus, compounds used in the compositions and methods of the present invention include all such combinations and variations.

In compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E, carbon atoms to which four non-identical substituents are bonded are asymmetric. Accordingly, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E may exist as enantiomers, diastereomers or a mixture thereof. The enantiomers and diastereomers may be separated by chromatographic or crystallization methods, or by other methods known in the art. The asymmetric carbon atom may be in one of two configurations, R or S, both of which are within the scope of the present invention. The presence of small amounts of the opposing enantiomer or diastereomer in the final purified product does not affect the therapeutic application of such compounds.

Compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E may be further treated to form pharmaceutically acceptable salts. Treatment of a compound of the invention with an acid or base may form, respectively, a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt, each as defined above. Various inorganic and organic acids and bases known in the art, including those described herein above, may be used to effect the conversion to the salt.

The present invention also relates to pharmaceutically acceptable isomers, hydrates, and solvates of compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E. Compounds of these formulae may also exist in various isomeric and tautomeric forms including pharmaceutically acceptable salts, hydrates and solvates of such isomers and tautomers.

This invention also encompasses prodrug derivatives of the compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E. The term “prodrug” refers to a derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, within the organism to release the parent drug. Prodrugs are variations or derivatives of the compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E of the present invention that have groups cleavable under metabolic conditions. Prodrugs become the compounds of the invention which are pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. An exemplary prodrug technology that may be suitably employed with the compounds of the present invention is the protease activated cancer therapy (PACT) technology described in detail within U.S. patent application Ser. No. 10/156,214 and PCT Application Publication No. WO 02/095007, both of which are incorporated herein by reference.

Synthesis of compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E may be achieved by reacting an acid chloride, obtained by reacting p-menthane-3-carboxylic acid with thionyl chloride, with the appropriate amine. As noted below, typically, the reaction is carried out in solution at room temperature in the presence of a hydrogen chloride receptor (e.g., sodium hydroxide).

The basic p-menthane structure is a chair-shaped molecule that can exist in cis or trans form. Substitution of the carboxyl or amide group into the 3-position gives rise to four configurational or geometric isomers depending upon whether the substitution is axially or equatorially into the cis or trans isomer, the four isomers are related as menthol is to neomenthol, isomenthol, and neoisomenthol.

In exemplary embodiments, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E are synthesized with a particular stereochemistry wherein the relative stereochemistry about the menthane ring is that of Menthol and/or wherein the absolute stereochemistry about the menthane ring is that of (−)-Menthol.

Synthetic methods for the preparation of exemplary Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E small-molecule Trp-p8 modulators of the present invention are presented herein in Examples 1-9.

Compositions Comprising Small-Molecule Trp-p8 Modulators

As discussed above, the present invention is directed to small-molecule Trp-p8 modulators, including Trp-p8 agonists and Trp-p8 antagonists that bind to and alter the activity of Trp-p8. Within certain embodiments, Trp-p8 modulators are agonists that are, in certain instances, capable of stimulating cation influx in, and toxicity of, a cell expressing the Trp-p8 channel protein. Within alternative embodiments, Trp-p8 modulators are antagonists of Trp-p8 activity that are capable of reducing the activity of Trp-p8 expressed in a cell. Thus, Trp-p8 modulators of the present invention will find utility in compositions, including pharmaceutical compositions, which are useful in the treatment of diseases associated with Trp-p8 expression. Suitable compositions, according to the present invention, comprise one or more Trp-p8 agonist of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E and/or one or more Trp-p8 antagonist of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E, as described above, in combination with one or more pharmaceutically acceptable carrier or excipient.

In one embodiment, the present invention provides small-molecule Trp-p8 modulators in combination with a pharmaceutically acceptable excipient such as sterile saline or other medium, water, gelatin, oil, etc., to form pharmaceutically acceptable compositions. The compositions and/or agonists may be administered alone or in combination with any convenient carrier, diluent, etc. and such administration may be provided in single or multiple dosages. Useful carriers include, but are not limited to, solid, semi-solid, or liquid medium including water and non-toxic organic solvents.

Pharmaceutical compositions of the present invention may be prepared by mixing one or more Trp-p8 agonist of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E with a pharmaceutically acceptable carrier or agent. Alternatively, pharmaceutical compositions may be prepared by mixing one or more Trp-p8 antagonist of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E with a pharmaceutically acceptable carrier or agent. In addition, pharmaceutical compositions may further include excipients, stabilizers, diluents and the like and may be provided in sustained release or timed release formulations. Acceptable carriers, agents, excipients, stabilizers, diluents and the like for therapeutic use are well known in the pharmaceutical field, and are described, for example, in “Remington's Pharmaceutical Sciences,” (Mack Publishing Co., ed. A. R. Gennaro, 1985), incorporated herein by reference. Such materials are nontoxic to the recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, acetate, and other organic acid salts, antioxidants such as ascorbic acid, low molecular weight peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulin, hydrophilic polymers such as serum albumin, gelatin, or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidinone, amino acids such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium and/or nonionic surfactants such as TWEEN, or polyethyleneglycol.

Within still further aspects, the compositions of the present invention comprise a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E formulated together with one or more cancer therapeutic agent. Alternatively, the compositions of the present invention comprise a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E independently formulated with one or more cancer therapeutic agent. That is, the compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E and the cancer therapeutic agent are separately formulated.

Suitable cancer therapeutic agents include, but are not limited to, antimitotic agents including, but not limited to, paclitaxel, vincristine, and etoposide; alkylating agents including, but not limited to, mechlorethamine, cyclophosphamide, and carmustine; antimetabolites including, but not limited to, methotrexate, gemcitabine, lometrexol, 5-fluorouracil, and 6-mercaptopurine; cytotoxic antibiotics including, but not limited to, doxorubicin, daunorubicin, bleomycin, mitomycin C, and streptozocin; platinum agents including, but not limited to, cisplatin and carboplatin; hormonal agents including, but not limited to, anti-estrogens such as tamoxifen and diethylstilbestrol as well as anti-androgens such as flutamide; antiangiogenesis agents; and farnesyl transferase inhibitors.

In certain aspects, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are administered in combination with a cancer therapeutic agent that is ineffective in stimulating Trp-p8-mediated cation influx.

In other aspects, compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are administered in combination with one or more additional Trp-p8 modulator including, but not limited to a compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E.

Depending upon the particular treatment regimen contemplated, pharmaceutical compositions of the present invention may be administered parenterally, topically, orally, or locally. In certain aspects, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In one embodiment, the present invention provides compositions for parenteral administration that comprise a compound of the present invention, dissolved or suspended in a carrier such as an aqueous carrier.

For solid formulations, compounds may be admixed with conventional nontoxic solid carriers such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For aerosol administration, compounds of the present invention may be supplied in finely divided form along with a nontoxic surfactant and propellant. Exemplary such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, actanoic, lauric, palmitic, stearic, linoleic, olesteric, and oleic acids.

Compositions of the present invention may be administered by injection, i.e. intravenously, intramuscularly, intracutaneously, subcutaneously, introaduodenally, or intraperitoneally. Alternatively, compositions may be administered by inhalation, such as intranasally, and may be administered transdermally, such as through a patch or the like.

It will be understood that the actual preferred formulation of compositions, including pharmaceutical compositions, will vary according to the mode of administration as well as the particular disease being treated. The optimal formulations and modes of administration will be routinely determined on a disease by disease and patient by patient basis by those of skill in the art.

Methods for Identifying and Characterizing the In Vitro and In Vivo Efficacy of Small-Molecule Modulators of Trp-p8

As discussed above, the present invention is directed to small-molecule Trp-p8 modulators, including agonists and antagonists of Trp-p8 activity. Disclosed herein are Trp-p8 modulators exemplified by the compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E described herein above. The present invention further contemplates that additional Trp-p8 modulators may also be suitably employed in the compositions and methods of the present invention.

Additional or alternative Trp-p8 agonists and antagonists may be identified by the methodology disclosed in the accompanying Examples. For instance, Trp-p8 agonists having efficacy in the treatment of disease(s) associated with Trp-p8 expression include small molecules that result in one or more of the following: (1) inhibit the growth or decrease the viability of a cell expressing Trp-p8; (2) stimulate calcium and/or other cation influx in a cell expressing Trp-p8; (3) induction of apoptosis and/or necrosis in a cell expressing Trp-p8; and/or (4) efficacy in one or more animal model systems of human disease. Trp-p8 antagonists having efficacy in the treatment of disease(s) associated with Trp-p8 expression include small molecules that result in one or more of the following: (1) protect Trp-p8 expressing cells from toxic effect of agonists in vitro model system (2) inhibit growth of and/or kill cancer cell line with endogenous Trp-p8 expression (3) are efficacious in one or more animal model systems of human disease.

Thus, within certain embodiments, the present invention provides methods for identifying Trp-p8 agonists comprising the step of contacting a Trp-p8 expressing cell with a candidate Trp-p8 agonist for a time and in an amount sufficient to inhibit the growth and/or decrease the viability of a Trp-p8 expressing cell, wherein the inhibited growth and/or reduced viability indicate that the candidate Trp-p8 agonist is capable of activating Trp-p8 expressed by the cell.

Other embodiments provide methods for identifying Trp-p8 agonists, comprising the step of contacting a Trp-p8 expressing cell with a candidate Trp-p8 agonist for a time and in an amount sufficient to induce influx of calcium and/or other cations into the cell, wherein increased cation influx is correlative of increased cellular toxicity.

Still further embodiments provide methods for identifying Trp-p8 agonists comprising the step of administering a candidate Trp-p8 agonist to an animal having one or more neoplastic cell that expresses Trp-p8 for a time and in an amount sufficient to inhibit the growth of and/or induce apoptosis and/or necrosis in the cell thereby increasing the survival of the animal, wherein any one or more of inhibition of cell growth, induction of apoptosis, induction of necrosis, and/or increased survival of the animal indicate efficacy of the Trp-p8 agonist.

The present invention provides methods for the identification of Trp-p8 antagonists in addition to the Trp-p8 antagonists disclosed herein. Such method include (1) in vitro assay systems for detecting the protection of Trp-p8 expressing cells from toxicity induced by Trp-p8 agonists; (2) in vitro and in vivo assay systems of detecting the inhibition of growth of a cancer cell and/or cancer cell line endogenously expressing Trp-p8; (3) in vivo animal model systems whereby one or more candidate Trp-p8 antagonist is administered to an animal having one or more neoplastic cell that expresses Trp-p8 for a time and in an amount sufficient to inhibit the growth of and/or induce apoptosis and/or necrosis in the cell thereby increasing the survival of the animal.

Methods for Use of Trp-p8 Modulators

Small-molecule Trp-p8 modulators of the present invention may be suitably employed in methods for modifying (i.e. activating or reducing) Trp-p8-mediated calcium influx in a cell and therapeutic methods for the treatment of one or more diseases associated with expression of Trp-p8. For example, and as noted above, it has been observed that abnormal Trp-p8 expression is associated with a neoplastic phenotype in a variety of cancerous tissues including breast, colon, lung, and prostate tissues. Tsavaler et al., Cancer Research, supra.

Thus, within certain embodiments are provided methods for activating Trp-p8-mediated calcium influx in a cell, such methods comprising the step of contacting the Trp-p8 expressing cell with an amount of a Trp-p8 agonist for a time sufficient to inhibit growth of the cell and/or to induce necrosis and/or apoptosis in the cell. Exemplary methods for activating Trp-p8 are provided within the Examples presented herein.

Other embodiments of the present invention provide therapeutic methods for the treatment of diseases associated with expression of Trp-p8, such methods comprising the step of administering to a mammal, typically a human, a therapeutically effective amount of a composition comprising a Trp-p8 agonist for a time sufficient to inhibit growth of the cell and/or to induce necrosis and/or apoptosis in the cell. As used herein, the phrase “therapeutically effective amount” refers to the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending upon the compound, the disease, and its severity and the age, weight, etc., of the mammal to be treated.

As used herein, the terms “treat”, “treating”, and “treatment” include: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be predisposed to the disease but does not yet experience any symptoms of the disease; (2) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e. causing regression of the disease or its clinical symptoms.

While the frequency and dosage of treatment regimens will vary depending upon such factors as the disease and patient treated, compositions comprising one or more compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are typically administered in the range of about 0.001 mg compound/kg body mass to about 1000 mg/kg. Typically, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage may be increased until optimal effectiveness is achieved.

In most instances, administration of a composition(s) of the present invention is achieved by any method that ensures systemic exposure to the compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E. Thus, compositions may be administered orally, parenterally, intraduodenally, and intranasally. Typically, such compositions comprise one or more such compound in combination with one or more pharmaceutically acceptable carrier or diluent, as described in further detail herein above.

Other embodiments of the present invention provide combination therapies wherein one or more compound of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E is administered in conjunction with one or more cancer therapeutic agent, as described in further detail herein above, such as an antimitotic agent, an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a platinum agent, a hormonal agent, and/or an antiandrogen. Still further embodiments of the present invention provide combination therapies wherein two or more compounds of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and/or Formula I-E are administered either simultaneously or sequentially to achieve the desired therapeutic outcome.

Thus, as used herein, the term “combination” means that at least two compounds can be delivered in a simultaneous manner, in combination therapy wherein the first compound is administered first, followed by the second compound, as well as wherein the second compound is delivered first, followed by the first compound. The desired result can be either a subjective relief of a symptom(s) or an objectively identifiable improvement in the recipient of the dosages.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1 Synthesis of Menthane-3-Carboxamide Compounds

This example discloses methodology for the synthesis of Menthane-3-carboxamide compounds.

Menthane-3-Carboxylic Acid

Water (300 ml) was placed in a 2-L Erlenmeyer flask with a large stir bar. Sulfuric acid (500 ml) was added carefully with stirring. The solution was allowed to cool to 75° C., and N-ethyl-p-menthane-3-carboxamide (62.5 g) was added. The temperature was maintained at 75° C. with a hot plate, and sodium nitrite (31 g) was added carefully. Two more 31 gram portions of NaNO₂ were added at 1-hour intervals, and the mixture was stirred overnight at 75° C.

The mixture was cooled to room temperature, diluted with ˜1 L of ice water, and extracted with ˜500 ml of ether. The ether layer was separated, washed with water, and extracted with 2×350 ml of 1M NaOH. The aqueous layer was made acidic with concentrated HCl and extracted with ether. The ether layer was dried with MgSO₄ and evaporated to give menthane-3-carboxylic acid (33.2 g, 61%) as a crystalline solid, =−50.3 deg (c=1, CHCl₃, 25° C.).

Menthane-3-Carbonyl Chloride

Menthane-3-carboxylic acid (54.35 g) was refluxed with 80 ml of thionyl chloride for 3 hours. The SOCl₂ was removed by distillation, and the acid chloride was distilled at 114-115° C. (8 Torr). (Lit. b.p. 84-85° C. at 3.5 Torr). Yield: 50 g (84%).

General Procedure for Preparation of Menthane-3-Carboxamides

To a stirred solution of 0.2 mmol of the amine in 1 ml of acetonitrile or NMP and 0.4 mmol of DIPEA was added 0.022 ml of menthane-3-carbonyl chloride. The reaction mixture was shaken for 3 hours. For less reactive amines, the mixture was heated (60° C.) and shaken for 24 hours. The product was purified from the crude reaction mixture by HPLC (40-95% gradient over 10 minutes using 0.05% TFA in CH₃CN and 0.05% TFA in H₂O) and evaporated to dryness.

Example 2 Synthesis of Dihydrobenzoimidazol Compounds of Formula I-E

This example discloses methodology for the synthesis of dihydrobenzoimidazol Trp-p8 modulators of Formula I-E.

4-Methoxy-2-fluoro-1-nitrobenzene

A 2 L round bottom flask equipped with a stir bar and reflux condenser was charged with acetonitrile (1 L), K₂CO₃ (263 g, 1.9 mol) and 4-hydroxy-2-fluoro-1-nitrobenzene (100 g, 0.64 mol). Methyl iodide (271 g, 1.9 mol) was added to the reaction mixture and heated at reflux temperatures with vigorous stirring for 5 h. The acetonitrile was removed and ethyl acetate (1 L) and H₂O (700 mL) were added. The heterogeneous mixture was transferred to a reparatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×200 mL). The organic phases were combined and washed with H₂O (2×500 mL), brine (500 mL), dried over MgSO₄, filtered and concentrated under reduced pressure to give the desired product as a slightly yellow solid (107 g, 98%).

[2-(5-Methoxy-2-nitrophenylamino)-ethyl]-carbamic acid tert-butyl ester

A 2 L flask equipped with a stir bar was charged with DMSO (800 mL), K₂CO₃ (161 g, 1.6 mol) and 4-methoxy-2-fluoro-1-nitrobenzene (100 g, 0.58 mol). Mono-N-Boc-1,2-diaminoethane (94 g, 0.55 mol) was added to the reaction mixture and stirred for 12 h at 60° C. The reaction mixture was triturated with ice cold water (1.2 L) and the resulting yellow precipitate was collected by vacuum filtration. The precipitate was washed several times with water (5×1 L) and dried on a high vacuum for 48 h to give the desired product as a bright yellow solid (178 g, 98%.)

[2-(5-Methoxy-2-Amino-phenylamino)-ethyl]-carbamic acid tert-butyl ester

A 2 L round bottom flask equipped with a stir bar was charged with a suspension of 20% Pd(OH)₂ (5 g) and 1,4-dioxane (800 mL). [2-(5-Methoxy-2-nitrophenylamino)-ethyl]-carbamic acid tert-butyl ester (100 g, 0.32 mol) was added to the suspension. The reaction mixture was hydrogenated (balloon) for 48 h (until the starting material had been consumed) followed by the addition of K₂CO₃ (100 g) was added to the mixture and stirred for an additional 12 h to remove traces of water. The suspension was filtered to remove the Pd(OH)₂ and K₂CO₃. The filtrate was used in the next step without further purification (yield not determined).

[2-(6-Methoxy-2-Oxo-2,3-dihydrobenzoimidazol-1-yl)-ethyl]-carbamic acid tert-butyl ester

The above solution was treated with an excess of carbonyl diimidazole (104 g, 0.64 mol) and heated at 90° C. for 4 h. The 1,4-dioxane was removed and the residue was triturated with water (1.5 L). The resulting precipitate was collected by vacuum filtration and washed several times with water (5×500 mL). The crude product was dried at 70° C. on the high vacuum for 12 h and used without further purification (66 g, 67% yield for 2 steps).

{2-[3-(2-Isopropyl-5-methylcyclohexanecarbonyl)-6-methoxy-2-oxo-2,3-dihydrobenzoimidazol-1-yl]-ethyl}-carbamic acid tert-butyl ester

A 2 L flask equipped with a stir bar was charged with [2-(2-oxo-2,3-dihydrobenzoimidazol-1-yl)-ethyl]-carbamic acid tert-butyl ester (40 g, 0.20 mol), DMAP (48 g, 0.39 mol) and CH₂Cl₂ (500 mL). Menthoyl chloride (40 g, 0.20 mol) was added drop wise over a 15 minute period and stirred at an ambient temperature for 4 h. The reaction mixture was quenched with 1N HCl and stirred for an additional 20 min. The heterogeneous mixture was transferred to a separation funnel where the aqueous phase was separated and re-extracted with CH₂Cl₂ (2×200 mL). The organic phases were combined and washed with 1N HCl (2×300 mL), H₂O (300 mL), saturated NaHCO₃ (aq) (2×300 mL), brine (300 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The crude product was dissolved in a minimal amount of CH₂Cl₂ and eluted through a plug of silica gel (10% hexane/ethyl acetate for elution) to furnish the desired product as a colorless solid (93 g, 96%).

TFA salt of 3-(2-Aminoethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-5-methoxy-1,3-dihydro-benzoimidazol-2-one (Compound #36)

A 500 mL round bottom flask was charged with {2-[3-(2-Isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydrobenzoimidazol-1-yl]-ethyl}-carbamic acid tert-butyl ester (90 g, 0.19 mol) and 95% TFA/H₂O (200 mL). The reaction was stirred for 2 h. and the TFA was removed under reduced pressure to give the crude product as a thick oil (which solidifies to form fragile foam upon standing under vacuum). The crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (79 g, 94%). MS (ESI) m/z 374 (M⁺+1).

TFA salt of 3-(2-Aminoethyl)-5-ethoxy-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-1,3-dihydro-benzoimidazol-2-one (Compound #38)

In a procedure similar to the synthesis of Compound #36, Compound #38 was prepared from 4-ethoxy-1-2-fluoro-1-nitrobenzene (prepared from ethyl bromide and 4-hydroxy-2-fluoro-1-nitrobenzene. MS (ESI) m/z 387 (M⁺+1).

TFA salt of 1-(2-Aminoethyl)-3-(2-isopropyl-5-methylcyclohexanecarbonyl)-1,3-dihydro-benzoimidazol-2-one (Compound #50)

In a procedure similar to the synthesis of Compound #36, Compound #50 was prepared from 2-fluoro-1-nitrobenzene. MS (ESI) m/z 344 (M⁺+1).

TFA salt of 3-(2-aminoethyl)-5-(3-hydroxypropoxy)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-1,3-dihydro-benzoimidazol-2-one (Compound #37)

In a procedure similar to the synthesis of Compound #36, Compound #37 was prepared from 4-(2-tert-butoxypropoxy)-2-fluoro-1-nitrobenzene (prepared from 1-bromo-3-tert-butoxy propane and 4-hydroxy-2-fluoro-1-nitrobenzene). MS (ESI) m/z 418 (M⁺+1).

TFA salt of 3-(2-aminoethyl)-5-(2-hydroxyethoxy)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-1,3-dihydro-benzoimidazol-2-one (Compound #40)

In a procedure similar to the synthesis of Compound #36, Compound #40 was prepared from 4-(2-tert-butoxyethoxy)-2-fluoro-1-nitrobenzene (prepared from 1-bromo-3-tert-butoxy ethane and 4-hydroxy-2-fluoro-1-nitrobenzene). MS (ESI) m/z 404 (M⁺+1).

TFA salt of 1-(2-Amino-2-(R)-methylethyl)-3-(2-isopropyl-5-methylcyclohexanecarbonyl)-1,3-dihydro-benzoimidazol-2-one (Compound #45)

In a procedure similar to the synthesis of Compound #36, Compound #45 was prepared from 2-fluoro-1-nitrobenzene and (2-amino-1-(R)-ethyl)carbamic acid tert-butyl ester. MS (ESI) m/z 358 (M⁺+1).

Example 3 Synthesis of Additional Dihydrobenzoimidazole Compounds of Formula I-E

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-p8 modulators of Formula I-E.

3-Fluoro-4-Nitrobenzoic Acid Methyl Ester

A 1 L round bottom flask equipped with a stir bar and reflux condenser was charged H₂SO₄ (4 mL), methanol (400 mL) and 3-fluoro-4-nitrobenzoic acid (10 g). The reaction mixture was heated at reflux temperatures with vigorous stirring for 18 h. The methanol was removed and the crude residue was triturated with hexane and concentrated to give a colorless solid (9.79 g) that was used without further purification.

3-(2-tent-Butyoxycarbonylaminoethylamino)-4-nitrobenzoic acid methyl ester

A 2 L flask equipped with a stir bar was charged with 1,4-dioxane (300 mL), DMF (40 mL), K₂CO₃ (10 g) and 3-fluoro-4-nitrobenzoic acid (9.7 g). Mono-N-Boc-1,2-diaminoethane (8.6 g) was added to the reaction mixture and stirred for 12 h at 60° C. The reaction mixture was concentrated and the residue was dissolved in CH₂Cl₂ (400 mL) and H₂O (500 mL). The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with CH₂Cl₂ (2×100 mL). The organic phases were combined and washed with H₂O (5×100 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to provide the title compound as an orange solid (14 g, 84%).

4-Amino-3-(2-tert-butoxycarbonylamino-ethylamino)-benzoic acid methyl ester

A 2 L round bottom flask equipped with a stir bar was charged with a suspension of 20% Pd(OH)₂ and 1,4-dioxane (400 mL). 4-Amino-3-(2-tert-butoxycarbonylamino-ethylamino)-benzoic acid methyl ester (14 g) was added to the suspension. The reaction mixture was hydrogenated (balloon) for 48 h (until the starting material had been consumed) followed by the addition of K₂CO₃ (100 g) was added to the mixture and stirred for an additional 12 h to remove traces of water. The suspension was filtered to remove the Pd(OH)₂ and K₂CO₃. The filtrate was used in the next step without further purification.

3-(2-tert-Butoxycarbonylaminoethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid methyl ester

The above solution was treated with an excess of carbonyl diimidazole (26.8 g, 4 eq.) and heated at 90° C. for 4 h. The 1,4-dioxane was removed and the residue was triturated with water (1.5 L). The resulting precipitate was collected by vacuum filtration and washed several times with water (5×500 mL). The crude product was dissolved in a minimal amount of CH₂Cl₂ and purified by flash chromatography on silica gel (10% methanol/CH₂Cl₂ for elution) to furnish the desired product as an off white solid (11.8 g, 85%).

3-(2-tert-Butoxycarbonylaminoethyl)-2-oxo-2,3-dihydro-1H-benzoimidzole-5-carboxylic acid

A 2 L flask equipped with a stir bar was charged with 1,4-dioxane (70 mL), 3-(2-tert-butoxycarbonylaminoethyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid methyl ester (10.4 g) and LiOH (3.7 g) dissolved in H₂O (300 mL). The reaction solution was stirred for 6 h at 65° C. The mixture was concentrated and the crude residue was dissolved in H₂O. The solution was neutralized with conc. HCl (aq.) and the resulting precipitate was collected by vacuum filtration. The solid was washed several times with H₂O and dried on the high vacuum overnight to provide the desired product as a white solid (8.66 g, 87%).

TFA salt of 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benzoimidazol-5-carboxylic acid

In a 100 mL reaction vessel equipped with stir bar was charged with THF (20 mL), DMAP (1.8 g) and 3-(2-tert-Butoxycarbonylaminoethyl)-2-oxo-2,3-dihydro-1H-benzoimidzole-5-carboxylic acid (4 g). The reaction mixture was cooled to 0° C. and treated with menthoyl chloride (2.9 g). The reaction mixture was allowed to warm to ambient temperature and concentrated. 1N HCl (aq) (50 mL) and CH₂Cl₂ (50 mL) was added. The heterogeneous mixture was transferred to a reparatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×100 mL). The organic phases were combined and washed with 1N HCl (2×50 mL), H₂O (50 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (4:1 CH₂Cl₂/THF for elution) provided the title compounds as colorless solid (3.2 g, 52%).

TFA salt of 3-(2-Amino-ethyl)-1-(2-isoprophyl-5-methyl-cyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benoimidazole-5-carboxylic acid amide (Compound #41)

In a 10 mL reaction vessel equipped with stir bar was charged with DMF (5 mL), 3-(2-amino-ethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benzoimidazol-5-carboxylic acid (1.5 g, 3.9 mmol), EDC (824 mg, 4.3 mmol), HOBt (581 mg, 4.3 mmol), DIEA (1.11 g, 8.6 mmol) and NH₄Cl (230 mg, 4.3 mmol). The reaction mixture was heated via microwave at 60° C. for 10 min and poured into a mixture of ethyl acetate (50 mL) and 1N HCl (50 mL). The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×50 mL). The organic phases were combined and washed with 1N HCl (100 mL), H₂O (2×100 mL), sat. NaHCO₃ (3×100 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was dissolved in 95% TFA/H₂O and stirred for 2 h and concentrated. The crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 10-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (910 mg, 61%). MS (ESI) m/z 387 (M⁺+1).

TFA salt of 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid (2-diethylamino-ethyl)amide (Compound #44)

In a procedure similar to the synthesis of Compound #41, Compound #44 was prepared from N¹,N¹-Diethylethan-1,2-diamine. MS (ESI) m/z 486 (M⁺+1).

TFA salt of 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid ethyl amide (Compound #47)

In a procedure similar to the synthesis of Compound #41, Compound #47 was prepared from ethyl amine. MS (ESI) m/z 415 (M⁺+1).

TFA salt of 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methylcyclohexanecarbonyl)-2-oxo-2,3-dihydro-1H-benzoimidazole-5-carboxylic acid pyridine-3-yl amide (Compound #48)

In a procedure similar to the synthesis of Compound #41 Compound #48 was prepared from pyridine-3-ylamine. MS (ESI) m/z 464 (M⁺+1).

Example 4 Synthesis of Additional Dihydrobenzoimidazole Compounds of Formula I-E

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-p8 modulators of Formula I-E.

[2-(5-Methyl-sulfanyl-2-nitro-phenylamino)-ethyl]-carbamic acid tert-butyl ester

In a 1 liter round bottom flask equipped with a stir bar was charged with DMSO (200 mL), K₂CO₃ (13 g, 0.10 mol) and 2,4-difluoro-1-nitrobenzene (5 g, 0.03 mol). The reaction mixture was treated with Mono-N-Boc-1,2-diaminoethane (5 g, 0.32 mol) and stirred at ambient temperature for 18 h. Sodium thiomethoxide (2.24 g, 0.03 mol) was added to the reaction mixture and stirred for 12 h at 60° C. The reaction mixture was cooled to 0° C. and triturated with water (800 mL) and the yellow precipitate that formed was collected by vacuum filtration. The precipitate was washed several times with water (5×500 mL) and dried on a high vacuum for 48 h to give the desired product as a bright yellow solid (8.7 g, 71%).

[2-(2-amino-5-Methyl-sulfanyl-phenylamino)-ethyl]-carbamic acid tert-butyl ester

In a 500 mL round bottom flash equipped with a stir bar was charged with MeOH (200 mL), [2-(5-methyl-sulfanyl-2-nitro-phenylamino)-ethyl]-carbamic acid tert-butyl ester (5 g, 0.02 mol) and NiCl₂ (19 g, 0.05 mol) and cooled to 0° C. NaBH₄ (1.7 g, 0.05 mol) was added (in four equal portions) to the reaction mixture over a 1 h period. Once the addition was complete the reaction mixture was stirred for an additional 2 h. Brine (100 mL) and ethyl acetate (200 mL) were added to the reaction mixture. The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×100 mL). The organic phases were combined and washed with H₂O (3×100 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure to give a black residue. The crude product was dissolved in 100 mL of CH₂Cl₂ and separated into two 100 mL round (50 mL in each) and both were concentrated under reduced pressure and used without further purification.

3-(2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methylsulfanyl-1,3-dihydro-benzoimidazol-2-one (Compound #42)

In a procedure similar to the synthesis of Compound #36, Compound #42 was prepared from the crude [2-(2-amino-5-Methyl-sulfanyl-phenylamino)-ethyl]-carbamic acid tert-butyl ester. MS (ESI) m/z 390.1 (M⁺+1).

3-2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methylsulfinyl-1,3-dihydro-benzoimidazol-2-one (Compound #43)

A 10 mL reaction flask was charged with 3-(2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methylsulfanyl-1,3-dihydro-benzoimidazol-2-one (Compound #42, 300 mg) and 1% TFA/DMSO (1 mL). Oxygen was bubbled through the reaction mixture for 20 min and sealed. The reaction mixture was stirred for 18 h and crude product was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (296 mg 94%). MS (ESI) m/z 406 (M⁺+1).

3-2-Amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methylsulfonyl-1,3-dihydro-benzoimidazol-2-one (Compound #39)

A 10 mL reaction vessel equipped with a stir bar was charged with 3-(2-amino-ethyl)-1-(2-isopropyl-5-methyl-cyclohexanecarbonyl)-5-methylsulfanyl-1,3-dihydro-benzoimidazol-2-one (Compound #42), Oxone (1 g) and 20% aqueous methanol (5 mL). The reaction mixture is titrated with sat. NaHCO₃ (aq) to a pH of ˜5. Reaction mixture is stirred for 1 h. The reaction mixture is filtered and concentrated. The crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 15-50% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a fluffy colorless solid (79 g, 94%). MS (ESI) m/z 422 (M⁺+1).

Example 5 Synthesis of Compounds of Formula I-B

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-p8 modulators of Formula I-A

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (4-methoxy-2-nitrophenyl)-amide

4-Methoxy-2-nitroaniline (5 g, 0.018 mol) was dissolved in pyridine (50) and treated with menthoyl chloride (3.57 g, 0.018 mol). The reaction mixture was heated to 50° C. and stirred vigorously for 6 h. The reaction mixture was cooled to room temperature and poured into a mixture of CH₂Cl₂ (100 mL) and 1N HCl (100 mL)). The heterogeneous mixture was transferred to a reparatory funnel where the aqueous phase was separated and re-extracted with CH₂Cl₂ (2×100 mL). The organic phases were combined and washed with 1N HCl (8×100 mL), H₂O (1×100 mL), 1N NaOH (2×100 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (20 to 50% ethyl acetate/hexane for elution) provided the title compounds as colorless solid (4.9 g, 83%).

2-Isopropyl-5-methyl-cyclohexanecarboxylic (2-amino-4-methoxyphenyl)-amide

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (4-methoxy-2-nitrophenyl)-amide (4.9 g) was dissolved in a suspension of 10% Pd—C (5 g) and THF (150 mL). The reaction mixture was hydrogenated over 20% Pd(OH)₂ for 48 h with a balloon. The reaction mixture was filtered and concentrated to give the desired compound in sufficient purity to use in the next reaction without further purification.

(1-{2-[2-Isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-5-methoxy-phenylcarbamoyl}-ethyl)carbamic acid tert-butyl ester

2-Isopropyl-5-methyl-cyclohexanecarboxylic (2-amino-4-methoxyphenyl)-amide (5 g, 0.016 mol), EDC (4.2 g, 0.022 mol), HOBt (2.97 g, 0.022 mol) and DIEA (8.53 g, 0.066 mol) were dissolved in DMF (50 mL) and stirred at 45° C. for 6 h. The reaction mixture was cooled to room temperature and poured into a mixture of ethyl acetate and 1N HCl (100 mL). The heterogeneous mixture was transferred to a reparatory funnel and the phases where separated. The aqueous phase re-extracted with ethyl acetate ( ) and the organic phases where combined, washed with 1N HCl (5×100 mL), H₂O (100 mL), saturated 1N NaOH (2×100 mL), brine (100 mL), dried (MgSO₄), filtered and concentrated to give a slightly yellow solid (7.5 g). A portion of the crude product (1.5 g) was purified by flash chromatography with silica gel (SiO₂, 30% ethyl acetate/hexane for elution) to give the desired product as a colorless solid (1.6 g).

TFA salt of 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid-[2-(2-aminopropionylamino)-4-methoxylphenyl]amide (Compound #1)

(1-{2-[2-Isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-5-methoxy-phenylcarbamoyl}-ethyl)carbamic acid tert-butyl ester (1 g) was dissolved in 95% TFA/H₂O and stirred for 1 h. The reaction mixture was concentrated and the crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (880 mg) MS (ESI) m/z 376 (M⁺+1).

TFA salt of 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid [2-(2-amino-ethylamino)-4-methylsulfanyl-phenyl]amide (Compound #4)

A 100 mL round bottom flask equipped with a stir bar containing crude [2-(2-amino-5-methyl-sulfanyl-phenylamino)-ethyl]-carbamic acid tert-butyl ester was charged with THF (50 g) and DMAP (1.8 g, 0.02 mol). The reaction mixture was cooled to 0° C. and menthoyl chloride (1.5 g, 0.008 mol) was added drop wise over a 5 min period. The reaction mixture was allowed to warm to ambient temperature and stirred for an additional 30 min. The crude product was dissolved in a minimal amount of CH₂Cl₂ and purified by flash chromatography on silica gel (10% hexane/ethyl acetate for elution) which resulted in a slightly yellow solid (1.76 g, 61%). The purified material was dissolved in 20 mL of 95% TFA/H₂O and stirred for 1 h and concentrated. The crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (1.41 g). MS (ESI) m/z 364 (M⁺+1)

[2-(4-Fluoro-2-nitro-benzoylamino)-ethyl]-carbamic acid tert-butyl ester

In a 100 mL round bottom flask equipped with a stir bar was charged with acetonitrile (40 mL), EDC (1.12 g, 5.9 mmol), HOBt (0.796 g, 5.9 mmol), DIEA (3.76 mL, 21.6 mmol) and Mono-N-Boc-1,2-diaminoethane (0.865 g, 5.4 mmol). The reaction mixture was stirred for ˜18 h and concentrated. The residue was dissolved in a mixture of ethyl acetate (50 mL) and 1N HCl (50 mL). The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×100 mL). The organic phases were combined and washed with 1N HCl (2×50 mL), H₂O (1×50 mL), sat NaHCO₃ (3×50 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (30% to 50% ethyl acetate/hexane for elution) provided the title compounds as slightly purple solid (1.12 g, 63%).

[2-(5-Methyl-sulfanyl-2-nitro-phenylamino)-ethyl]-carbamic acid tert-butyl ester

In a 10 reaction vessel equipped with a stir bar was charged with DMF (5 mL), NaSMe (0.162 g, 2.3 mmol) and [2-(4-fluoro-2-nitro-benzoylamino)-ethyl]-carbamic acid tert-butyl ester (0.757 g, 2.3 mmol). The reaction mixture stirred at ambient temperature for 2 h and poured into a mixture of ethyl acetate (20 mL) and H₂O (25 mL). The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×10 mL). The organic phases were combined and washed with 1N HCl (2×10 mL), H₂O (1×10 mL), sat NaHCO₃ (2×10 mL), brine (10 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (30% to 50% ethyl acetate/hexane for elution) provided the title compounds as slightly yellow solid (500 mg, 61%).

N-(2-Amino-ethyl-2-[(2-isopropyll-5-methyl-cyclohexanecarbonyl)-amino-4-methylsulfanyl-benzamide (Compound #2)

In a procedure similar to the synthesis of Compound #42, Compound #2 was prepared from the [2-(5-Methyl-sulfanyl-2-nitro-phenylamino)-ethyl]-carbamic acid tert-butyl ester. MS (ESI) m/z 392 (M⁺+1).

Example 6 Synthesis of Compounds of Formula I-B

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-p8 modulators of Formula I-B.

2-(5-Amino-3-phenyl-pyrazol-1-yl)-ethanol

Benzoylacetonitrile (25 g, 0.17 mol) was suspended in a mixture of 125 mL reagent grade anhydrous alcohol and 20 mL glacial acetic acid. 2-Hydroxyethylhydrazine (14.4 g, 1.1 equiv) dissolved in 35 mL alcohol was added all at once. The mixture was heated at reflux for 4 h, cooled, water was added to make 500 mL total volume and the solution was chilled in a refrigerator overnight. Crystals were filtered cold on a Buchner funnel, washed with cold water and dried on high vacuum to give the desired product (27.2 g, 79%).

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid [2-(2-hydroxy-ethyl)-5-phenyl-2H-pyrzol-3-]-amide

The 2-(5-amino-3-phenyl-pyrazol-1-yl)-ethanol (87.3 g, 0.43 mol) was suspended in a mixture of dichloromethane (500 mL) and pyridine (40 mL) and chilled in an ice bath. Menthoyl chloride (100 g, 1.15 equiv) was dissolved in dichloromethane (200 mL) and added drop wise from an addition funnel protected by a CaCl₂ drying tube. After the 45 min required for complete addition, the ice bath was removed and stirring continued for 3 h. 1M HCl (aq, 200 mL)) was added was added and the phases were separated. The organic phases was re-extracted with 1M HCl (aq, 100 mL). 1M HCl was again added and the dichloromethane was removed under reduced pressure resulting in profuse precipitation. The precipitate was collect by vacuum filtration and the solid was washed with water several times. The solid residue was triturated with 400 mL 1:1 ether/hexanes (rapid stirring for 2 h). The solid was filtered on a Buchner funnel and washed with hexanes. After air drying overnight, further drying was effected on high vacuum for 24 h to give a colorless solid (144.4 g).

Methanesulfonic acid-2-{5-[2-isopropyl-5-methyl-cyclohexanecarboyl)-amino]-3-phenyl-pyrzol-1-yl}ethyl ester

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid [2-(2-hydroxy-ethyl)-5-phenyl-2H-pyrzol-3-]-amide (140 g, 0.38 mol) was suspended in CH₂Cl₂ (500 mL) and pyridine (47 mL, 1.5 equiv) was added followed by methanesulfonyl chloride (44 mL, 1.5 equiv) at ice bath temperature. The solution was allowed to warm to room temperature and was stirred for an additional 12 h. Water (500 mL) was added and the mixture was stirred for 0.5 h. Dichloromethane was removed by evaporation leaving a precipitate of 1t. yellow granular chunks. Decantation was followed by treatment with an additional 500 mL water and decantation again. A final 500 mL quantity of water was used to transfer the solid to a Buchner funnel where it was suction dried (yield not determined).

2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-azido-ethyl)-5-phenyl-2H-pyrzol-3-yl]-amide

The crude mesylate (0.38 mol) was dissolved in DMSO (500 mL) with sodium azide (37 g, 1.5 equiv). The mixture was heated to 70° C. for 6 h. Upon cooling, water (1 L) and ethyl acetate (500 mL) were added and the mixture was shaken in a reparatory funnel. The layers were separated and the organic layer was washed sequentially with 200 mL quantities of water, saturated NaHCO₃, and brine. The organic layer was dried with Na₂SO₄, decanted, and the solvent removed on the rotovap. Yield was not determined because the product was not quite free of solvent before moving to the next step.

2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-amino-ethyl)-5-phenyl-2H-pyrzol-3-yl]-amide

Crude azido compound was dissolved in 500 mL reagent grade absolute alcohol and treated with 5 g activated carbon. This was stirred for several hours and filtered through Celite. Approximately 300 mL solvent was removed on the rotovap and replaced with fresh solvent. 10% Pd—C (4.8 g, ˜50%-wt. H₂O) was added, and a steady stream of hydrogen was maintained over the reaction mixture with rapid stirring for 24 h. Hydrogen was disconnected and conc. HCl (32 mL) was added slowly. After filtration through Celite, the filtrate was concentrated on the rotovap resulting in profuse precipitation. Still wet, diisopropyl ether was added to the residue and the suspension was stirred rapidly for 0.5 h. The solid was filtered into a Buchner funnel and washed with diethyl ether. Air dried white powder was produced.

Yield: 108.6 g (71% over three steps).

TFA salt of 2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-amino-ethyl)-5-phenyl-2H-pyrzol-3-yl]-amide (Compound #16)

Conversion to the trifluoroacetate salt: the solid was neutralized and partitioned in a separatory funnel by shaking with 500 mL ether and 150 mL 2 N NaOH When the solid was completely dissolved, the layers were separated and the organic phase was dried with Na₂CO₃. Decantation and mixing with 23 mL trifluoroacetic acid was followed by evaporation of solvent and drying on high vacuum. The foam was crushed and triturated with 300 mL hexanes (rapid stirring for 3 h) which, upon filtration, produced a white powder containing much less ether. Solvent was finally removed completely by heating in a round bottom flask at 80° C. for 6 h.

TFA salt of 2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrzol-3-yl]-amide (Compound #14)

In a procedure similar to the synthesis of Compound #16, Compound #14 was prepared from 2-furoylacetonitrile and 2-hydroxyethylhydrazine. This material was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. MS (ESI) m/z 344 (M⁺+1).

TFA salt of 2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl]-amide (Compound #17)

In a procedure similar to the synthesis of Compound #16, Compound #17 was prepared from 1-methyl-1H-pyrrole-2-carbaldehyde and 2-hydroxyethylhydrazine. This material was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. MS (ESI) m/z 372 (M⁺+1).

TFA salt of 2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl]-amide (Compound #15)

In a procedure similar to the synthesis of Compound #16, Compound #15 was prepared from 2-benzoylacetonitrile and (2-diazenyl-ethyl)-carbamic acid tert-butyl ester. This material was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. MS (ESI) m/z 383 (M⁺+1).

TFA salt of 2-Isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl]-amide (Compound #18)

In a procedure similar to the synthesis of Compound #16, Compound #18 was prepared from 2-benzoylacetonitrile and (3-diazenyl-propyl)-carbamic acid tert-butyl ester. This material was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. MS (ESI) m/z 383 (M⁺+1).

Example 7 Synthesis of Compounds of Formula I-C

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-n8 modulators of Formula I-C

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (2-hydroxy-2-phenyl-ethyl)-amide

A 500 mL round bottom flask equipped with stir bar was charged with CH₂Cl₂ (200 mL), DIEA (28 g, 0.219 mol), and 2-Amino-1-phenyl-ethanol (10 g, 0.073 mol) and cooled to 0° C. Menthoyl chloride (14.8 g, 0.073 mol) was added drop wise over a 15 min period. Once the addition was complete the reaction was allowed to warm to ambient temperature and stirred 2 h. CH₂Cl₂ (100 mL) and 1N HCl (100 mL) was added to the reaction mixture and stirred for an additional 20 min. The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with CH₂Cl₂ (2×100 mL). The organic phases were combined and washed with 1N HCl (8×100 mL), H₂O (1×100 mL), 1N NaOH (2×100 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was eluted through a plug of silica gel (50% ethyl acetate/hexane for elution) provided the title compounds as colorless solid (18.8 g, 85%).

TFA salt of 2-Isopropyl-5-methyl-cyclohexanecarboxylic [2-(2-amino-4-ethoxy)-2-phenyl-ethyl]-amide (Compound #30)

A 500 mL round bottom flask equipped with stir bar was charged with anhydrous THF (200 mL) and 2-isopropyl-5-methyl-cyclohexanecarboxylic acid (2-hydroxy-2-phenyl-ethyl)-amide (10 g, 0.03 mol). NaH (0.87 g, 0.04 mol) was added in one portion and stirred for 10 min (until H₂ stopped being produced). 1-Bromoethyl-2-amine hydrogen bromide (6.74 g, 0.033 mol) and NaH (0.87 g, 0.036 mol) were added to the reaction mixture and stirred for 2 h. An additional equivalent of NaH (0.87 g, 0.036 mol) was added and stirred an additional 2 h. The excess NaH was quenched by pouring the reaction mixture onto ice. Ethyl acetate (200 mL) and H₂O were added and stirred for 20 min. The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×100 mL). The organic phases were combined and washed with H₂O (1×100 mL), brine (100 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The crude product was dissolved in 30% acetonitrile/H₂O and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 30-60% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a light fluffy colorless solid (9.4 g, 62%). MS (ESI) m/z 347 (M⁺+1).

TFA salt of 2-Isopropyl-5-methyl-cyclohexanecarboxylic [2-(3-amino-4-propoxy)-2-phenyl-ethyl]-amide (Compound #31)

In a procedure similar to the synthesis of Compound #30, Compound #31 was prepared from 2-isopropyl-5-methyl-cyclohexanecarboxylic acid (2-hydroxy-2-phenyl-ethyl)-amide and 1-Bromopropyl-3-amine hydrogen bromide. This material was purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 40-60% acetonitrile/H₂O (with 0.1% TFA) gradient. MS (ESI) m/z 361 (M⁺+1).

Example 8 Synthesis of Additional Dihydrobenzoimidazole Compounds of Formula I-D

This example discloses methodology for the synthesis of dihydrobenzoimidazole Trp-p8 modulators of Formula I-D

2-Isopropyl-5-methyl-cyclohexanecarboylic acid [2-(2-bromo-phenyl)-ethyl]-amide

A 100 mL round bottom flask equipped with a stir bar was charged with CH₂Cl₂ (30 mL), 2-bromo-phenethylamine (1.0 g, 5.00 mmol) and triethylamine (684 μl, 5.05 mmol). The reaction solution was treated with menthoyl chloride (1.02 g, 5.05 mmol) in one portion and stirred at ambient temperature for 30 minutes. The reaction was diluted with CH₂Cl₂ (50 mL) and washed with water (3×100 mL). Organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a thick oil (1.8 g). Product was used for next step without purification.

TFA salt of 2-Isopropyl-5-methyl-cyclohexanecarboylic acid {2-[2-(2-amino-ethylamino)-phenyl]-ethyl}-amide (Compound #33)

A 25 mL microwave reaction vessel equipped with a stir bar was charged with neat diaminoethane (10 mL), 2-isopropyl-5-methyl-cyclohexanecarboylic acid [2-(2-bromo-phenyl)-ethyl]-amide (1.5 g, 4.1 mmol) and Cu powder (390 mg, 6.147 mmol, 1.5 eq.). The reaction vessel was subjected to microwave at 180° C. for 40 minutes. The reaction mixture was transferred to a round bottom flask and concentrated. The residue was dissolved in DMSO (1 mL) and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 10-40% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a colorless solid (1 g, 52%) (MS (ESI) m/z 346 (M⁺+1).

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid [2-(2-cyano-phenyl)-ethyl]-amide

A 20 mL microwave reaction vessel equipped with a stir bar was charged with 2-isopropyl-5-methyl-cyclohexanecarboylic acid [2-(2-bromo-phenyl)-ethyl]-amide (1.54 g, 4.2 mmol), CuCN (0.60 g, 6.4 mmol) and NMP (10 mL). The reaction vessel was subjected to microwave at 180° C. for 40 minutes. The reaction mixture was transferred to a round bottom flask and concentrated. The residue to purified by flash chromatography on silica gel (10% ethyl acetate/hexane for elution) to give a colorless solid (1.25 g, 81%).

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid [2-2-aminomethyl-phenyl)-ethyl]-amide (Compound #34)

A 100 mL round bottom flask equipped with a stir bar was charged with 2-isopropyl-5-methyl-cyclohexanecarboxylic acid [2-(2-cyano-phenyl)-ethyl]-amide (1.25 g, 4.0 mmol) and methanol (50 mL). NiCl₂ (1.14 g, 8.8 mmoles) and NaBH₄ (0.64 g, 16.8 mmol). NaBH₄ was added in small portion over a 30 min. period and stirred for 1 h. NaBH₄ (0.20 g) was added and reaction mixture and stirred an additional 20 minutes. The reaction mixture was passed through a cake of celite and concentrated under reduced pressure. The black residue was dissolved in a minimal amount of acetonitrile and passed through a C18 silica gel cartridge and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 10-40% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a colorless solid (1.1 g). MS (ESI) m/z 317 (M⁺+1).

Example 9 Synthesis of Additional Compounds of Formula I-C

This example discloses methodology for the synthesis of Trp-p8 modulators of Formula I-C

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (2-oxo-2-phenyl-ethyl)-amide

A 20 mL round bottom flask equipped with a stir bar was charge with 2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (2-hydroxy-2-phenyl-ethyl)-amide (100 mg, 0.33 mmol) and acetic acid (1 mL). A solution of CrO₃ (36 mg, 0.363 mmoles, 1.1 eq) in acetic acid (500 μl) and water (100 μl) was slowly added to the reaction mixture. The reaction mixture was stirred at ambient temperature for 15 min and diluted with ethyl acetate (30 mL) and saturated NaHCO₃ (aq.) (30 mL). The heterogeneous mixture was transferred to a reparatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×10 mL). The organic phases were combined and washed with sat NaHCO₃ (3×10 mL), H₂O (10 mL), brine (10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (30% to 50% ethyl acetate/hexane for elution) provided the title compounds as white solid (92 mg, 93%).

2-Isopropyl-5-methyl-cyclohexanecarboxylic acid (2-amino-2-phenyl-ethyl)-amide

A 25 mL microwave reaction vessel equipped with a stir bar was charged with 2-isopropyl-5-methyl-cyclohexanecarboxylic acid (2-oxo-2-phenyl-ethyl)-amide (80 mg) and ammonia (1.5 mL, 7 M in methanol). A drop of acetic acid and NaCNBH₃ (20 mg) were added to the reaction mixture and subjected to microwave at 80° C. for 80 min. The residue was taken up in ethyl acetate (30 mL) and saturated NaHCO₃ (aq.) (30 mL). The heterogeneous mixture was transferred to a reparatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×10 mL). The organic phases were combined and washed with sat NaHCO₃ (3×10 mL), H₂O (10 mL), brine (10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a solid (75 mg).

(1-{2-[(2-Isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-1-phenyl-ethylcarbamoyl}-ethyl)-carbamic acid tert-butyl ester

A 15 mL reaction vessel equipped with a stir bar was charged with THF (15 mL), Boc-(R)-alanine (52 mg, 0.273 mmoles), HOBt (37.87 mg, 0.273 mmoles), EDCI (53 mg, 0.273 mmoles) and TEA (37 μl, 0.273 mmoles). The reaction mixture was stirred for 15 min. whereupon 2-isopropyl-5-methyl-cyclohexanecarboxylic acid (2-amino-2-phenyl-ethyl)-amide (75 mg, 0.248 mmol) was added and stirred for an additional 3 h. Ethyl acetate (10 mL) and H₂O (10 mL) were added to the reaction mixture. The heterogeneous mixture was transferred to a separatory funnel where the aqueous phase was separated and re-extracted with ethyl acetate (2×10 mL). The organic phases were combined and washed with 1N HCl (2×10 mL), H₂O (1×10 mL), sat NaHCO₃ (3×10 mL), brine (10 mL), dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (20% ethyl acetate/hexane for elution) provided the title compounds as colorless solid (30 mg).

TFA salt of 2-Isopropyl-5-methyl-cyclohexancarboxylic acid [2-(2-amino-propionylamino)-2-phenyl-ethyl]-amide (Compound #28)

A 5 mL round bottom flask equipped with a stir bar was charged with 10% TFA/CH₂Cl₂ and (1-{2-[(2-isopropyl-5-methyl-cyclohexanecarbonyl)-amino]-1-phenyl-ethylcarbamoyl}-ethyl)-carbamic acid tert-butyl ester (30 mg) and stirred for 1 h. The TFA was removed under reduced pressure and the residue was dissolved in 30% acetonitrile/H₂O (with 0.1% TFA) and purified by preparative HPLC (Ultro 120 (10 um) C18Q) using a 10-40% acetonitrile/H₂O (with 0.1% TFA) gradient. The pure fractions were combined, concentrated and lyophilized to give a colorless solid (17.7 mg) (MS (ESI) m/z 374 (M⁺+1).

Example 10 Expression of Trp-p8 in CHO Cells

Human Trp-p8 transfected CHO cells (referred to herein as CHO/Trp-p8) were generated for use in experiments of the present invention. Expression of Trp-p8 polypeptide in this transfectant and the absence of any endogenous expression in the non-transfected CHO was confirmed by western blot and immunofluorescence using a Trp-p8 specific antibody (GS2.20) as well as the calcium flux assay with Icilin (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one) and menthol (2-isopropyl-5-methyl-cyclohexanol). Non-transfected CHO cells were used to establish the specificity of the effects of the compounds observed with CHO/Trp-p8.

Example 11 Trp-p8-mediated Decrease in Cell Viability following Exposure of CHO/Trp-p8 Cells with Candidate Trp-p8 Agonist Compounds at 37° C.

This Example discloses an ATP viability assay suitable for screening for effective Trp-p8 agonists. The ATP viability assay described herein employs CHO cells expressing an exogenous Trp-p8 cDNA. This example further establishes that Trp-p8 agonists of the present invention are effective in decreasing the viability of Trp-p8 expressing cells.

The concentration of intracellular ATP declines very rapidly when metabolically active cells undergo necrosis and/or apoptosis. The ATP concentration and consequently the relative cell viability can be measured by established methods using commercially available reagents. In the agonist screening methodology disclosed herein, a compound that specifically decreases the viability of CHO/Trp-p8 cells is referred to as an agonist.

As a primary screen for efficacy and specificity for agonists, both the non-transfected CHO and CHO/Trp-p8 cells were exposed to 1 or 10 μM of test compounds in 1% dimethylsulfoxide (DMSO) or 1% DMSO (control) in a 96-well black walled, black-bottomed, cell-culture treated plate. DMSO was the solvent for all of the compounds tested. After 24-26 hours at 37° C., the cells were lysed and ATP concentration determined via a chemiluminescence assay using a commercially available reagent kit—Cell Titer-Glo (Promega; Madison, Wis.). Relative viability (%), expressed as the ATP level in cells treated with compounds expressed as a percentage of ATP levels in cells treated with the DMSO alone, was a measure of the agonist activity of the candidate compound—the lower the % viability, the more potent the Trp-p8 agonist. EC₅₀ values were determined for the most active candidate Trp-p8 agonists at 37° C. by measuring viability at 8-10 agonist concentrations. (EC₅₀ is defined herein as the agonist concentration at which there is a 50% reduction in relative cell viability).

Exemplary Trp-p8 Agonists of Formula I, Formula I-A, Formula I-B, Formula I-C, Formula I-D, and Formula I-E, that were efficacious in the ATP viability assay are presented herein in Tables 1-5. EC50 data is designated as follows: A=<0.020 uM; B=0.021-0.050 uM; C=0.051-0.10 uM.

In Tables 1-5, the structures provided are of the form:

Wherein chemical names are provided for X and/or Y. Where names are provided for “X/Y”, the names are inclusive of the nitrogen group.

Viability of CHO/Trp-p8 cells following treatment with exemplary Trp-p8 agonists is presented in FIG. 1.

TABLE 1 Exemplary Compounds of Formula I-A Compound # Structure EC50 X 1

A 2-(2-amino-propionylamino)-4-methoxy- phenyl 2

A N-(2-Amino-ethyl)-2-amino-5-methylsulfanyl- phenyl 3

A 1-(2-amino-ethoxy)-naphthalen-2-yl 4

A 2-(2-amino-ethylamino)-4-methylsulfanyl- phenyl 5

A N-(2-Amino-ethyl)-5-methoxy-benzamide 6

B 2-(2-amino-butyrylamino)-4-methoxy-phenyl 7

B 2-(2-amino-3-hydroxy-propionylamino)-4- methoxy-phenyl 8

B 3-(2-amino-ethylamino)-naphthalen-2-yl 9

B N-(2-Amino-ethyl)-2-amino-benzamide 10

B 2-(2-amino-3-hydroxy-propionylamino)-4- methoxy-phenyl 11

C 2-(2-amino-acetylamino)-phenyl 12

C 2-(2-amino-3-hydroxy-butyrylamino)-4- methoxy-phenylamide 13

C 2-(2-amino-acetylamino)-4-methoxy-phenyl

TABLE 2 Exemplary Compounds of Formula I-B Object ID Structure EC50 X 14

A 2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrazol-3- yl 15

A 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl 16

A 2-(2-amino-ethyl)-5-phenyl-2H-pyrazol-3-yl 17

A 2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)- 2H-pyrazol-3-yl 18

A 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl 19

B 2-(2-amino-ethyl)-5-(4-amino-phenyl)-2H- pyrazol-3-yl 20

B 2-(2-amino-ethyl)-5-(4-hydroxy-phenyl)-2H- pyrazol-3-yl 21

B 2-(2-methylamino-ethyl)-5-phenyl-2H- pyrazol-3-yl 22

B 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl 23

B 2-(2-amino-ethyl)-5-(3-cyano-phenyl)-2H- pyrazol-3-yl 24

B 2-(2-amino-ethyl)-5-(3-methoxy-phenyl)-2H- pyrazol-3-yl 25

C 4-{1-(2-Amino-ethyl)-1H-pyrazol-3-yl}- benzoic acid methyl ester 26

C 2-(2-amino-ethyl)-5-(3-amino-phenyl)-2H- pyrazol-3-yl 27

C 2-(2-amino-ethyl)-5-(3-hydroxy-phenyl)-2H- pyrazol-3-yl

TABLE 3 Exemplary Compounds of Formula I-C Compound # Structure EC50 X 28

A 2-(2-amino-propionylamino)-2-phenyl-ethyl 29

A 2-(2-amino-ethoxy)-2-phenyl-ethyl 30

A 2-(2-amino-ethoxy)-2-phenyl-ethyl 31

B 2-(3-amino-propoxy)-2-phenyl-ethyl 32

C 2-(2-amino-ethylamino)-2-phenyl-ethyl

TABLE 4 Exemplary Compounds of Formula I-D Compound # Structure EC50 X 33

A 2-[2-(2-amino-ethylamino)-phenyl]-ethyl 34

A 2-(2-aminomethyl-phenyl)-ethyl 35

B 2-[(2-amino-acetyl)-phenyl-amino]-ethyl

TABLE 5 Exemplary Compounds of Formula I-E Compound # Structure EC50 X/Y 36

A 3-(2-Amino-ethyl)-5-methoxy-1,3-dihydro- benzoimidazol-2-one 37

A 3-(2-Amino-ethyl)-5-(3-hydroxy-propoxy)-1,3- dihydro-benzoimidazol-2-one 38

A 3-(2-Amino-ethyl)-5-ethoxy-1,3-dihydro- benzoimidazol-2-one 39

A 3-(2-Amino-ethyl)-5-methanesulfonyl-1,3- dihydro-benzoimidazol-2-one 40

A 3-(2-Amino-ethyl)-5-(2-hydroxy-ethoxy)-1,3- dihydro-benzoimidazol-2-one 41

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid amide 42

A 3-(2-Amino-ethyl)-5-methylsulfanyl-1,3- dihydro-benzoimidazol-2-one 43

A 3-(2-Amino-ethyl)-5-methanesulfinyl-1,3- dihydro-benzoimidazol-2-one 44

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid (2- diethylamino-ethyl)-amide 45

A 3-(2-Amino-propyl)-2,3-dihydro- benzoimidazol-2-one 46

A [3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazol-5-yloxy]-acetonitrile 47

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid ethylamide 48

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid pyridin-3- ylamide 49

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid (2- methoxy-ethyl)-amide 50

A 1-(2-Amino-ethyl)-1,3-dihydro-benzoimidazol- 2-one 51

A 1-(2-Amino-ethyl)-1,3-dihydro-naphtho[2,3- d]imidazol-2-one 52

A 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole- 5-carboxylic acid (2-hydroxy-ethyl)-amide 53

A 3-(2-Amino-ethyl)-5-propoxy-1,3-dihydro- benzoimidazol-2- one 54

B 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5- c]pyridin-2-one 55

B 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-4-carboxylic acid (2- diethylamino-ethyl)-amide 56

B 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid pyridin-4- ylamide 57

B 3-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5- b]pyridin-2-one 58

B 1-(3-Amino-propyl)-1,3-dihydro- benzoimidazol-2-one 59

B 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid phenylamide 60

B 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid [2-(2- hydroxy-ethoxy)-ethyl]-amide 61

B 1-(2-Amino-ethyl)-5-trifluoromethyl-1,3- dihydro-benzoimidazol-2-one 62

B 1-(2-Amino-ethyl)-1,3-dihydro-imidazo[4,5- c]pyridin-2-one 63

B 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid benzylamide 64

B 3-(2-Amino-ethyl)-5-(morpholine-4-carbonyl)- 1,3-dihydro-benzoimidazol-2-one 65

B 3-(2-Amino-ethyl)-5-(2-oxo-2-phenyl-ethoxy)- 1,3-dihydro-benzoimidazol-2-one 66

B 3-(2-methylamino-ethyl)-1,3-dihydro- benzoimidazol-2-one 67

C 3-(2-Amino-ethyl)-5-butoxy-1,3-dihydro- benzoimidazol-2-one 68

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid methyl- phenyl-amide 69

C 4-[3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carbonyl]-piperazine-1- carboxylic acid ethyl ester 70

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid diethylamide 71

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid phenethyl- amide 72

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid (2-hydroxy- 1-hydroxymethyl-2-phenyl-ethyl)-amide 73

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid carbamoylmethyl-amide 74

C 3-(2-Amino-ethyl)-2-oxo-2,3-dihydro-1H- benzoimidazole-5-carboxylic acid (2-hydroxy- 1-hydroxymethyl-ethyl)-amide 75

C 3-(2-Amino-ethyl)-5-benzyloxy-1,3-dihydro- benzoimidazol-2-one 76

C 1-(4-Amino-butyl)-1,3-dihydro-benzoimidazol- 2-one

Example 12 Screen and Characterization of Trp-p8 Agonist Compounds by Measuring Calcium Influx in CHO/Trp-p8 cells at 37° C.

This example discloses a CHO/Trp-p8-based calcium influx assay used to further assess the activity of candidate Trp-p8 agonists of the present invention.

Calcium influx was measured using a Flexstation Microplate Fluorescence Plate Reader (Molecular Devices; Sunnyvale, Calif.). A typical assay for calcium flux was performed as follows. Cells in DMEM/Ham's F-12 based medium, typically at a density of 30,000 cells/well/100 μl, were plated in a 96-well black-walled, clear bottomed tissue culture plate (Greiner Bio-one) and incubated for 16-20 hours at 37° C. Cells in each well were incubated for one hour at 37° C. with a Fura2-AM Fluorescent Dye/Pluronic F-27 mixture (Molecular Probes; Eugene, Oreg.) and dissolved in the medium containing Probenecid. Typical final concentrations were: 5-8 μM of Fura2-AM, 0.01% Pluronic F-27, and 2.5 mM Probenecid (an anion exchange inhibitor that reduces transport of the hydrolyzed dye from inside the cell thereby minimizing loss of dye during the experiment). After one hour, cells were washed in a buffered solution (20 mM HEPES and Hanks Balanced Salt Solution with 1.26 mM CaCl₂), pH 7.4 containing Probenecid at a final concentration of 2.5 mM and pre-incubated for at least 30 minutes at the assay temperature of 37° C.

Typically, the above described HEPES/HBSS-based buffer containing either no additional calcium or with calcium to increase the concentration to 2 mM and various concentrations of compounds (at 5-times the final concentrations) were added to each well using the robotic multi-channel pipettor. The compounds were preincubated at 37° C. for at least 30 minutes before performing the assay (at 37° C.). Signals were read with dual excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm with a cut-off filter at 495 nm. The signal was reported as the ratio of emission when excited at 340 nm to the emission when excited at 380 nm [Relative Fluorescence Units (RFU)]. Ionomycin was routinely used as a positive control.

In the case of the agonist assay, the compounds at different concentrations were added to the dye-loaded cells (as described above). The increase in RFU was a measure of potency of the compound as an agonist. Exemplary results are presented in FIG. 2.

Example 13 Increase in Apoptosis following Exposure of CHO/Trp-p8 Cells with Trp-p8 Agonist Compounds at 37° C.

This example discloses the effectiveness of Trp-p8 agonist compounds in inducing apoptosis in Trp-p8 expressing cells.

An Annexin V/Propidium Iodide (PI) flow cytometry assay was used to provide additional insights into the mechanism of cell death induced by Trp-p8 agonist compounds. Annexin V staining detects translocation of Phosphatidylserine to the outer layer of plasma membrane, an event characteristic of apoptosis, while PI staining indicates dead cells with compromised membranes.

Cells were treated with compounds in 1% DMSO or with a 1% DMSO (control) for 24-26 hours at 37° C. The cells were briefly trypsinized under controlled conditions and stained with an Annexin V/PI reagent kit following the methodology provided by the supplier (e.g., Southern Biotech; Birmingham, Ala.). Exemplary results are presented in FIG. 3.

Example 14 In Vitro Screen using a Cell Viability Assay for Trp-p8 Antagonist Compounds Based Upon Protection of Trp-p8-Expressing Cells from Toxic Agonist Compounds

This example discloses an assay system for identifying and characterizing candidate Trp-p8 antagonist compounds.

Trp-p8 antagonists were identified by employing a cell viability assay with CHO/Trp-p8 cells at 37° C. (see Example 11) with the following modification. Within the context of the present invention, compounds that protect CHO/Trp-p8 cells from the toxic effect of a control agonist thereby maintaining the viability of the CHO/Trp-p8 cell exposed to a Trp-p8 agonist is defined as antagonist. As a primary screen for antagonists, CHO/Trp-p8 cells were exposed to 10 μM of test compounds in 1% dimethylsulfoxide (DMSO) or 1% DMSO plus a toxic concentration of a control agonist. The relative viability at 10 μM, determined as described in Example 11, was a measure of the potential of the compound as a Trp-p8 antagonist—the higher the viability, the more potent the antagonist. Exemplary results are presented in FIG. 4.

Example 15 In Vitro Screen using a Calcium Flux Assay for Trp-p8 Antagonist Compounds Based upon their Abilities to Suppress the Calcium Influx Induced by Trp-p8 Agonists in CHO/Trp-p8 Cells

This example discloses an in vitro assay system employed to further screen and characterize candidate Trp-p8 antagonists.

Trp-p8 antagonists were also screened and characterized using a calcium flux assay at 37° C. as described in Example 12 with the following two distinctions: (1) the compound was pre-mixed with the control agonist or only the control agonist is added to the cells and suppression of the response to the agonist is a measure of the potency of the compound as an antagonist and (2) the compound, at different concentrations, was added to the cells followed by addition of the control agonist after 2-3 minutes and the suppression of response induced by agonist was a measure of potency of the compound as an antagonist. Exemplary results are presented in FIG. 5.

Example 16 An Animal Model System for Assaying the In vivo Efficacy of Candidate Trp-p8 Agonists and Antagonists for the Treatment of Cancer

This Example provides an animal model system suitable for determining the in vivo efficacy of candidate Trp-p8 modulators—including both agonists and antagonists.

Human prostate cancer xenografts expressing Trp-p8 (LuCaP, from Dr. Robert Vessella's lab in University of Washington—as assessed by in situ hybridization, quantitative polymerase chain reaction, and immunohistochemistry using a protein specific rabbit polyclonal antibody, T-904), as well as cell lines engineered to express Trp-p8, including CHO (Chinese Hamster Ovary) and EL-4 (Mouse Thymoma) cell lines, were used to establish tumor models in mice. Trp-p8 expression in the transfectants was confirmed by western blots and immunofluorescence using a Trp-p8 specific antibody (GS 2.20) as well as by response to known agonists in a calcium influx functional assay. In addition, the transfected cell lines were susceptible to killing by Trp-p8 agonists as evident from the ATP viability and apoptosis assays (as described herein in Examples 11 and 13).

A tumor model in mice was established by subcutaneously injecting CHO/Trp-p8 cells in SCID mice. Trp-p8 expression in tumors excised from these mice was confirmed by RT-PCR, immunohistochemistry, and western blot analysis. Further tumor model development is carried out using the human prostate cancer xenografts described above in athymic nude or SCID mice and using an EL4/Trp-p8 transfectant in normal mice. Prostate xenografts from other sources and other cell lines that may be engineered to express Trp-p8 are also potential candidates for building more model systems.

Based on results from in vitro and in vivo evaluations, a set of Trp-p8 agonists will be chosen to determine efficacy in mice. The in vitro evaluations would include potency in cell killing assay, aqueous solubility, plasma binding study and metabolic stability (potential for a compound to be metabolized by liver as determined by using hepatocytes and/or mouse microsomes). The in vivo evaluations would include pharmacokinetics and toxicity studies. The chosen compounds will be administered to mice with Trp-p8 expressing tumors by different routes [oral, intravenous, intraperitoneal, subcutaneous, intramuscular]. Tumor reduction and survival of these mice will be evaluated at different dosages of these compounds. The compound most effective in fighting tumor will be chosen for further investigations

Example 17 Experimental Characterization of Several Exemplary Compounds

This Example discloses the experimental characterization and results of several exemplary small-molecule Trp-p8 modulators of Formula I, designated Compound I, II, III and IV. Their chemical formulas and molecular weight are summarized in Table 6.

TABLE 6 Chemical Formulas and Molecular Weights Compound I II III IV Chemical C₂₁H₃₁N₃O₃ C₂₀H₂₉N₃O2 C₂₂H₃₂N₄O C₂₁H₃₄N₂O₂ Formula Molecular 373 343 368 346 Weight In Vitro Activity

As summarized in Table 8, the compounds demonstrated a high degree of potency and specificity towards killing cells that express Trp-p8. Typically, >1000× higher concentrations of compound were required to kill cells lacking Trp-p8, compared to cells that express Trp-p8. Compounds II, III, and IV showed similar activity in this assay, while Compound I was approximately 3 times more potent.

TABLE 8 Results of ATP viability assay for several preferred compounds Compound I II III IV CHO/Trp-p8 EC₅₀ 0.003 0.01 0.01 0.01 (uM) Parent CHO EC₅₀ >10 >10 >10 >10 (uM) In Vivo Activity

As illustrated in FIGS. 9A and 9B, Compounds I, II, and III produced prolonged exposure after a single oral administration in both rodents (FIG. 9A) and Beagle dogs (FIG. 9B). Compared to mice, rats require approximately twice the oral dose (based on body weight) to achieve comparable exposure, and dogs require less than a third. Consistent with the sustained plasma levels (t½˜9 h), a single oral dose affords a prolonged durable response in the CHO/Trp-p8 xenograft model.

As illustrated in FIGS. 10A and 10B, oral dosing of these compounds afforded durable responses in the CHO/Trp-p8 xenograft model. Substantial inhibition of tumor growth was seen after a single dose as low as 10 mg/kg, and no significant toxicity was evident at 100 mg/kg; a therapeutic window of >10×.

As illustrated in FIG. 7, compounds of Formula I generated substantially briefer exposure via a single intraperitoneal injection as compared to oral administration. As illustrated in FIGS. 8A and 8B, intraperitoneal injection of these compounds in mice results in briefer responses in the CHO/Trp-p8 xenograft model and appear less durable after cessation of IP dosing.

To demonstrate that efficacy is mediated by Trp-p8, FIG. 11 illustrates the evaluation of Compound I in the matched CHO (Trp-p8-) model. Consistent with the proposed mechanism of action, Compound I did not show significant efficacy at 100 mg/kg in this model; a dose 10 times higher than an efficacious dose in the analogous CHO/Trp-p8 model.

As illustrated in FIG. 12, the LuCaP model appeared to respond comparably or arguably better than the CHO/Trp-p8 model. CHO/Trp-p8 is a fast-growing tumor; treatment with Compound I attenuated growth, but did not cause regression. In contrast, LuCaP is a slower-growing tumor; treatment caused statistically significant regression, as well as growth-inhibition. In this instance, the LuCAP xenograft model exhibited levels of Trp-p8 comparable to the CHO/Trp-p8 model, as measured by immunohistochemistry of tumor tissues excised from the mice.

The highest dose orally administered to mice, 100 mg/kg, did not result in significant toxicity for any of the compounds. Since a single oral dose of 10 mg/kg of Compound I produced significant efficacy in the CHO/Trp-p8 xenograft model, a therapeutic window of >10 is achievable with Compounds of Formula I. This has been expanded upon by toxicology experiments in rats, where the compounds could be administered at higher dose levels. In toxicology studies performed in rats, oral doses of 250 mg/kg did not induce any observable toxic effects. Single Doses of 500 mg/kg and 1000 mg/kg resulted in mild to moderate toxicity, but the MTD was not reached. These data, representing the minimum therapeutic windows achievable with Compounds of Formula I, are summarized in Table 7.

TABLE 7 Comparable Multiple of lowest Rat oral dose Observed degree mouse efficacious oral dose (mg/kg) of toxicity oral dose (mg/kg) in mice 250 None 125 12.5 500 Mild 250 25 1000 Moderate 500 50

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims. 

What is claimed is:
 1. A compound of Formula I-B:

wherein R₁ is selected from the group consisting of H, alkyl, heteroalkyl, aryl, and arylalkyl; R_(2a) is selected from the group consisting of aryl, alkyl, heteroalkyl, and arylalkyl; R₃ is selected from the group consisting of alkyl, heteroalkyl, and arylalkyl; R₄ is selected from the group consisting of H, alkyl, heteroalkyl, and arylalkyl; or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof wherein R₁ is H.
 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof wherein R₃ is alkyl, heteroalkyl or arylalkyl, each of which has 1, 2, 3, 4, 5, or 6 carbons.
 4. The compound of claim 3 or a pharmaceutically acceptable salt thereof wherein R₃ is selected from methylene, ethylene, propylene, and butylene.
 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof wherein R₄ is selected from H and methyl.
 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof wherein R_(2a) is a group selected from phenyl, furan, methylpyrrole, methylbenzoate, aminophenyl, hydroxyphenyl, cyanophenyl, and methoxyphenyl.
 7. The compound of claim 1 or a pharmaceutically acceptable salt thereof wherein the moiety:

is selected from the group consisting of 2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-amino-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(4-hydroxy-phenyl)-2H-pyrazol-3-yl, 2-(2-methylamino-ethyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-cyano-phenyl)-2H-pyrazol-3-yl, 2-(2-amino-ethyl)-5-(3-methoxy-phenyl)-2H-pyrazol-3-yl, 4-{1-(2-Amino-ethyl)-1H-pyrazol-3-yl}-benzoic acid methyl ester, 2-(2-amino-ethyl)-5-(3-amino-phenyl)-2H-pyrazol-3-yl, and 2-(2-amino-ethyl)-5-(3-hydroxy-phenyl)-2H-pyrazol-3-yl.
 8. The compound of claim 7 wherein said compound is 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-furan-2-yl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-phenyl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(1-methyl-1H-pyrrol-2-yl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(4-amino-phenyl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(4-hydroxy-phenyl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-methylamino-ethyl)-5-phenyl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-propyl)-5-phenyl-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[, 2-(2-amino-ethyl)-5-(3-cyano-phenyl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(3-methoxy-phenyl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[4-{1-(2-amino-ethyl)-1H-pyrazol-3-yl}-benzoic acid methyl ester]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(3-amino-phenyl)-2H-pyrazol-3-yl]-amide, 2-isopropyl-5-methyl-cyclohexane carboxylic acid-[2-(2-amino-ethyl)-5-(3-hydroxy-phenyl)-2H-pyrazol-3-yl]-amide or a pharmaceutically acceptable salt thereof.
 9. The compound of claim 8 in isolated or purified form.
 10. A pharmaceutical composition comprising a compound of claim 1, and combinations thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 